Methods and compositions for gray leaf spot resistance in corn

ABSTRACT

The present invention relates to the field of plant breeding. More specifically, the present invention includes a method of using haploid plants for genetic mapping of traits of interest such as disease resistance. Further, the invention includes a method for breeding corn plants containing quantitative trait loci (QTL) that are associated with resistance to Gray Leaf Spot, a fungal disease associated with  Cercospora  spp.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/015,338, filed Aug. 30, 2013, now U.S. Pat. No. 8,779,232, which is acontinuation of U.S. patent application Ser. No. 13/590,669, filed Aug.21, 2012, abandoned, which claims priority to U.S. patent applicationSer. No. 12/201,008, filed Aug. 29, 2008, now U.S. Pat. No. 8,273,944,which claims the benefit of U.S. Provisional Patent Application No.60/966,706, filed Aug. 29, 2007, all of which are incorporated herein byreference in their entireties.

This application is a continuation of U.S. patent application Ser. No.14/015,338, filed Aug. 30, 2013, which is a continuation of U.S. patentapplication Ser. No. 13/590,669, filed Aug. 21, 2012, which claimspriority to U.S. patent application Ser. No. 12/201,008, filed Aug. 29,2008, which claims the benefit of U.S. Provisional Patent ApplicationNo. 60/966,706, filed Aug. 29, 2007, all of which are incorporatedherein by reference in their entireties.

INCORPORATION OF SEQUENCE LISTING

A sequence listing is contained in the file named“46_(—)25(54886_(—)003_US).txt” which is 2432213 bytes (measured inMS-Windows) and was created on Aug. 11, 2012, and comprising 1,361nucleotide sequences and is electronically filed herewith and isincorporated herein by reference.

FIELD OF INVENTION

The present invention relates to the field of plant breeding. Morespecifically, the present invention includes a method of using haploidplants for genetic mapping of traits such as disease resistance.Further, the invention includes a method for breeding corn plantscontaining quantitative trait loci (QTL) that are associated withresistance to gray leaf spot, a fungal disease associated withCercospora spp.

BACKGROUND OF INVENTION

The present invention provides methods and compositions forintrogressing disease resistance loci in corn. GLS is a global problemand, in addition to prevalence in Africa, Central America and SouthAmerica, it has spread across most of the U.S. Corn Belt over the past10-15 years. The fungus overwinters in field debris and requiresmoisture, usually in the form of heavy fogs, dews, or rains, to spreadits spores and infect corn. Increasing pervasiveness has been linked tono-till practices which promote retention of fungi, such as Cercosporazea (CZ), in the soil (Paul et al., Phytopathology 95:388-396 (2005)).Symptoms include a rectangular necrotic lesion which can coalesce tolarger affected regions and symptoms usually appear later in the growingseason. GLS in corn elicits an increased allocation of plant resourcesto damaged leaf tissue, leading to elevated risk for root and stalkrots, which ultimately results in even greater crop losses (Ward et al.,1999; Saghai-Maroof et al, Theor. Appl. Genet. 93:539-546 (1996)).Yield-loss associated with GLS can be high if the symptoms are heavy andappear early, with reported losses exceeding 50% (Ward et al., 1999).Recent work has identified there are at least two sister species of CZ,as well as potentially other isolates of Cercospora, capable of causingGLS (Carson et al., Maydica 51:89-92 (2006); Carson et al, Plant Dis.86:1088-109 (2002)). Genomic regions on maize Chromosomes 1, 2, 3, 4, 5,6, 7, and 8 have been associated with GLS using RFLP, AFLP and SSRmarkers (U.S. Pat. No. 5,574,210; Lehmensiek, et al., TAG, (2001);Clements, et al. Phytopathology (2000); Gorden et al. Crop Science(2004); Bubeck, et al., Crop Science, (1993); Saghai-Maroof et al.,Theor. Appl. Genet (1996)). Certain genomic regions, molecular markers,and QTL associated with GLS resistance have also been reported (WO2008/042185 A2).

Breeding for corn plants resistant to GLS can be greatly facilitated bythe use of marker-assisted selection. Of the classes of genetic markers,single nucleotide polymorphisms (SNPs) have characteristics which makethem preferential to other genetic markers in detecting, selecting for,and introgressing disease resistance in a corn plant. SNPs are preferredbecause technologies are available for automated, high-throughputscreening of SNP markers, which can decrease the time to select for andintrogress disease resistance in corn plants. Further, SNP markers areideal because the likelihood that a particular SNP allele is derivedfrom independent origins in the extant population of a particularspecies is very low. As such, SNP markers are useful for tracking andassisting introgression of disease resistance alleles, particularly inthe case of disease resistance haplotypes.

SUMMARY OF THE INVENTION

Various methods and compositions for identifying and obtaining cornplants with resistance to Gray Leaf Spot (GLS) are provided herein. Incertain embodiments, a method of identifying a corn plant comprising atleast one allele associated with Gray Leaf Spot (GLS) resistance allelein a corn plant comprising: a) genotyping at least one corn plant withat least one nucleic acid marker selected from the group consisting ofSEQ ID NOs:1-62, 64-70, 72-156, 158-172, 174-187, 189-377, 379, 380,382-409, 411-459, 461-1233, 1360 and 1361, and b) selecting at least onecorn plant comprising an allele of at least one of said markersassociated with Gray Leaf Spot (GLS) resistance is provided. In certainembodiments of the methods, at least one corn plant genotyped in step(a) and/or the at least one corn plant selected in step (b) is a cornplant from a population generated by a cross. In embodiments where thepopulation is generated by a cross, the cross can be effected bymechanical emasculation, chemical sterilization, or geneticsterilization of a pollen acceptor. In certain embodiments of themethods, genotyping is effected in step (a) by determining the allelicstate of at least one of said corn genomic DNA markers. In certainembodiments of the methods, the selected one or more corn plants canexhibit at least partial resistance to a GLS-inducing fungus or at leastsubstantial resistance to a GLS-inducing fungus. In certain embodimentsof the methods, the population can be generated by a cross of at leastone Gray Leaf Spot (GLS) resistant corn plant with at least one GrayLeaf Spot (GLS) sensitive corn plant. In certain embodiments of themethods, the population can be a segregating population or a haploidbreeding population. In certain embodiments of the methods, the crosscan be a back cross of at least one Gray Leaf Spot (GLS) resistant cornplant with at least one Gray Leaf Spot (GLS) sensitive corn plant tointrogress GLS resistance into a corn germplasm.

Also provided herein are corn plants obtained by any of theaforementioned methods of identifying corn plants that comprise allelesof genetic loci associated with Gray Leaf Spot resistance. In certainembodiments, a corn plant obtained by any of these aforementionedmethods can comprise at least one allele of a nucleic acid markerselected from the group consisting of SEQ ID NOs: 1-62, 64-70, 72-156,158-172, 174-187, 189-377, 379, 380, 382-409, 411-459, 461-1233 and SEQID NOs: 1360 and 1361, wherein said allele is associated with Gray LeafSpot (GLS) resistance. In certain embodiments, a corn plant obtained byany of these aforementioned methods can exhibit at least partialresistance to a GLS-inducing fungus or at least substantial resistanceto a GLS-inducing fungus. In certain embodiments, a corn plant obtainedby any of these aforementioned methods can be a haploid corn plant. Incertain embodiments, a corn plant obtained by any of the aforementionedmethods and comprising at least one of the alleles can comprise at leastone transgenic trait. In such embodiments, the transgenic trait can beherbicide tolerance and/or pest resistance. In embodiments where thecorn plant obtained is herbicide tolerant, herbicide tolerance can beselected from the group consisting of glyphosate, dicamba, glufosinate,sulfonylurea, bromoxynil and norflurazon herbicide tolerance.

In certain embodiments, methods of introgressing a Gray Leaf Spot (GLS)resistance QTL allele into a corn plant comprising: a) screening apopulation with at least one nucleic acid marker to determine if one ormore corn plants from the population comprise(s) an allele of saidmarker associated with a Gray Leaf Spot (GLS) resistance QTL selectedfrom the group consisting of QTL numbers 1-9, 14-33, 35, 38-42, 44-52,54-61, 63-71, 73-79, 81-92, 95-96, 99-106, 108-117, and 119-178 asprovided in FIG. 1; and b) selecting from said population at least onecorn plant comprising an allele of said marker associated with a GrayLeaf Spot (GLS) resistance are provided. In certain embodiments of themethods, at least one of the markers can be located within 5 cM, 2 cM,or 1 cM of at least one of the Gray Leaf Spot (GLS) resistance QTL. Incertain embodiments of the methods, at least one of the markers canexhibit a LOD score of greater than 4.0 with at least one of said GrayLeaf Spot (GLS) resistance QTL. In certain embodiments of the methods,the population can be generated by a cross of at least one Gray LeafSpot (GLS) resistant corn plant with at least one Gray Leaf Spot (GLS)sensitive corn plant. In certain embodiments of the methods, thepopulation can be a haploid breeding population. In certain embodimentsof the methods, the nucleic acid marker is selected from the groupconsisting of SEQ ID NOs: 858, 860, 862, 866, 875, 877, 881, 882, 883,and 1360.

Also provided herein are corn plants obtained by any of theaforementioned methods of identifying corn plants that comprise a GrayLeaf Spot resistance QTL. In certain embodiments, a corn plant obtainedby any of these aforementioned methods can comprise a Gray Leaf Spot(GLS) resistance QTL selected from the group consisting of QTL numbers1-9, 14-33, 35, 38-42, 44-52, 54-61, 63-71, 73-79, 81-92, 95-96, 99-106,108-117, and 119-178 as provided in FIG. 1. In certain embodiments, acorn plant obtained by any of these aforementioned methods can exhibitat least partial resistance to a GLS-inducing fungus or at leastsubstantial resistance to a GLS-inducing fungus. In certain embodiments,a corn plant obtained by any of these aforementioned methods can be ahaploid corn plant. In certain embodiments, a corn plant obtained by anyof the aforementioned methods and comprising at least one of the QTL cancomprise at least one transgenic trait. In such embodiments, thetransgenic trait can be herbicide tolerance and/or pest resistance. Inembodiments where the corn plant obtained is herbicide tolerant,herbicide tolerance can be selected from the group consisting ofglyphosate, dicamba, glufosinate, sulfonylurea, bromoxynil andnorflurazon herbicide tolerance.

Also provided herein are isolated nucleic acid markers for identifyingpolymorphisms in corn DNA. These isolated nucleic acids can be used in avariety of applications, including but not limited to, theidentification of corn plants that comprise alleles of genetic lociassociated with Gray Leaf Spot resistance. In certain embodiments, anisolated nucleic acid molecule for detecting a molecular markerrepresenting a polymorphism in corn DNA, wherein the nucleic acidmolecule comprises at least 15 nucleotides that include or areimmediately adjacent to said polymorphism, wherein said nucleic acidmolecule is at least 90 percent identical to a sequence of the samenumber of consecutive nucleotides in either strand of DNA that includeor are immediately adjacent to said polymorphism, and wherein saidmolecular marker is selected from the group consisting of SEQ ID NOs:1-26, 28-62, 64-70, 72-120, 122-140, 142-156, 158-172, 174, 176,178-187, 189-219, 221-223, 225-233, 235-247, 249-251, 253-377, 379, 380,382-409, 411-439, 441-459, 461-478, 481-532, 534-581, 583-584, 586-638,640-720, 722-726, 728-732, 734-745, 747-767, 769-772, 774-939, 941-1052,1055-1121, 1123-1185, 1187-1233, 1304 through SEQ ID NO: 1331, 1360, and1361. In certain embodiments, the molecular marker is selected from thegroup consisting of SEQ ID NOs: 858, 860, 862, 866, 875, 877, 881, 882,883, and 1360. In certain embodiments, the isolated nucleic acid furthercomprises a detectable label or provides for incorporation of adetectable label. In such embodiments that comprise or provide forincorporation of a detectable label, the detectable label is selectedfrom the group consisting of an isotope, a fluorophore, an oxidant, areductant, a nucleotide and a hapten. In certain embodiments, thedetectable label is added to the nucleic acid by a chemical reaction oris incorporated by an enzymatic reaction. In certain embodiments, theisolated nucleic acid molecule comprises at least 16 or 17 nucleotidesthat include or are immediately adjacent to the polymorphism. In otherembodiments, the nucleic acid molecule comprises at least 18 nucleotidesthat include or are immediately adjacent to the polymorphism orcomprises at least 20 nucleotides that include or are immediatelyadjacent to the polymorphism. In certain embodiments, the isolatednucleic acid molecule hybridizes to at least one allele of the molecularmarker under stringent hybridization conditions.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and form a part ofthe specification, illustrate the embodiments of the present inventionand together with the description, serve to explain the principles ofthe invention.

In the drawings:

FIG. 1. Markers associated with GLS resistance from association mappingstudies. “*” indicates a single nucleotide deletion.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The definitions and methods provided herein define the present inventionand guide those of ordinary skill in the art in the practice of thepresent invention. Unless otherwise noted, terms are to be understoodaccording to conventional usage by those of ordinary skill in therelevant art. Definitions of common terms in molecular biology may alsobe found in Alberts et al., Molecular Biology of The Cell, 3^(rd)Edition, Garland Publishing, Inc.: New York, 1994; Rieger et al.,Glossary of Genetics: Classical and Molecular, 5th Edition,Springer-Verlag: New York, 1991; and Lewin, Genes V, Oxford UniversityPress New York, 1994. The nomenclature for DNA bases as set forth at 37CFR §1.822 is used.

As used herein, a “locus” is a fixed position on a chromosome and mayrepresent a single nucleotide, a few nucleotides or a large number ofnucleotides in a genomic region.

As used herein, “polymorphism” means the presence of one or morevariations of a nucleic acid sequence at one or more loci in apopulation of one or more individuals. The variation may comprise but isnot limited to, one or more base changes, the insertion of one or morenucleotides or the deletion of one or more nucleotides. A polymorphismincludes a single nucleotide polymorphism (SNP), a simple sequencerepeat (SSR) and indels, which are insertions and deletions. Apolymorphism may arise from random processes in nucleic acidreplication, through mutagenesis, as a result of mobile genomicelements, from copy number variation and during the process of meiosis,such as unequal crossing over, genome duplication and chromosome breaksand fusions. The variation can be commonly found or may exist at lowfrequency within a population, the former having greater utility ingeneral plant breeding and the later may be associated with rare butimportant phenotypic variation.

As used herein, “marker” means a detectable characteristic that can beused to discriminate between organisms. Examples of such characteristicsmay include genetic markers, protein composition, protein levels, oilcomposition, oil levels, carbohydrate composition, carbohydrate levels,fatty acid composition, fatty acid levels, amino acid composition, aminoacid levels, biopolymers, pharmaceuticals, starch composition, starchlevels, fermentable starch, fermentation yield, fermentation efficiency,energy yield, secondary compounds, metabolites, morphologicalcharacteristics, and agronomic characteristics.

As used herein, “genetic marker” means polymorphic nucleic acid sequenceor nucleic acid feature. A “polymorphism” is a variation amongindividuals in sequence, particularly in DNA sequence, or feature, suchas a transcriptional profile or methylation pattern. Usefulpolymorphisms include single nucleotide polymorphisms (SNPs), insertionsor deletions in DNA sequence (Indels), simple sequence repeats of DNAsequence (SSRs) a restriction fragment length polymorphism, a haplotype,and a tag SNP. A genetic marker, a gene, a DNA-derived sequence, aRNA-derived sequence, a promoter, a 5′ untranslated region of a gene, a3′ untranslated region of a gene, microRNA, siRNA, a QTL, a satellitemarker, a transgene, mRNA, ds mRNA, a transcriptional profile, and amethylation pattern may comprise polymorphisms.

As used herein, “marker assay” means a method for detecting apolymorphism at a particular locus using a particular method, e.g.measurement of at least one phenotype (such as seed color, flower color,or other visually detectable trait), restriction fragment lengthpolymorphism (RFLP), single base extension, electrophoresis, sequencealignment, allelic specific oligonucleotide hybridization (ASO), randomamplified polymorphic DNA (RAPD), microarray-based technologies, andnucleic acid sequencing technologies, etc.

As used herein, the phrase “immediately adjacent”, when used to describea nucleic acid molecule that hybridizes to DNA containing apolymorphism, refers to a nucleic acid that hybridizes to DNA sequencesthat directly abut the polymorphic nucleotide base position. Forexample, a nucleic acid molecule that can be used in a single baseextension assay is “immediately adjacent” to the polymorphism.

As used herein, “interrogation position” refers to a physical positionon a solid support that can be queried to obtain genotyping data for oneor more predetermined genomic polymorphisms.

As used herein, “consensus sequence” refers to a constructed DNAsequence which identifies SNP and Indel polymorphisms in alleles at alocus. Consensus sequence can be based on either strand of DNA at thelocus and states the nucleotide base of either one of each SNP in thelocus and the nucleotide bases of all Indels in the locus. Thus,although a consensus sequence may not be a copy of an actual DNAsequence, a consensus sequence is useful for precisely designing primersand probes for actual polymorphisms in the locus.

As used herein, the term “single nucleotide polymorphism,” also referredto by the abbreviation “SNP,” means a polymorphism at a single sitewherein said polymorphism constitutes a single base pair change, aninsertion of one or more base pairs, or a deletion of one or more basepairs.

As used herein, “genotype” means the genetic component of the phenotypeand it can be indirectly characterized using markers or directlycharacterized by nucleic acid sequencing. Suitable markers include aphenotypic character, a metabolic profile, a genetic marker, or someother type of marker. A genotype may constitute an allele for at leastone genetic marker locus or a haplotype for at least one haplotypewindow. In some embodiments, a genotype may represent a single locus andin others it may represent a genome-wide set of loci. In anotherembodiment, the genotype can reflect the sequence of a portion of achromosome, an entire chromosome, a portion of the genome, and theentire genome.

As used herein, the term “haplotype” means a chromosomal region within ahaplotype window defined by at least one polymorphic molecular marker.The unique marker fingerprint combinations in each haplotype windowdefine individual haplotypes for that window. Further, changes in ahaplotype, brought about by recombination for example, may result in themodification of a haplotype so that it comprises only a portion of theoriginal (parental) haplotype operably linked to the trait, for example,via physical linkage to a gene, QTL, or transgene. Any such change in ahaplotype would be included in our definition of what constitutes ahaplotype so long as the functional integrity of that genomic region isunchanged or improved.

As used herein, the term “haplotype window” means a chromosomal regionthat is established by statistical analyses known to those of skill inthe art and is in linkage disequilibrium. Thus, identity by statebetween two inbred individuals (or two gametes) at one or more molecularmarker loci located within this region is taken as evidence ofidentity-by-descent of the entire region. Each haplotype window includesat least one polymorphic molecular marker. Haplotype windows can bemapped along each chromosome in the genome. Haplotype windows are notfixed per se and, given the ever-increasing density of molecularmarkers, this invention anticipates the number and size of haplotypewindows to evolve, with the number of windows increasing and theirrespective sizes decreasing, thus resulting in an ever-increasing degreeconfidence in ascertaining identity by descent based on the identity bystate at the marker loci.

As used herein, a plant referred to as “haploid” has a single set(genome) of chromosomes and the reduced number of chromosomes (n) in thehaploid plant is equal to that of the gamete.

As used herein, a plant referred to as “doubled haploid” is developed bydoubling the haploid set of chromosomes. A plant or seed that isobtained from a doubled haploid plant that is selfed any number ofgenerations may still be identified as a doubled haploid plant. Adoubled haploid plant is considered a homozygous plant. A plant isconsidered to be doubled haploid if it is fertile, even if the entirevegetative part of the plant does not consist of the cells with thedoubled set of chromosomes; that is, a plant will be considered doubledhaploid if it contains viable gametes, even if it is chimeric.

As used herein, a plant referred to as “diploid” has two sets (genomes)of chromosomes and the chromosome number (2n) is equal to that of thezygote.

As used herein, the term “plant” includes whole plants, plant organs(i.e., leaves, stems, roots, etc.), seeds, and plant cells and progenyof the same. “Plant cell” includes without limitation seeds, suspensioncultures, embryos, meristematic regions, callus tissue, leaves, shoots,gametophytes, sporophytes, pollen, and microspores.

As used herein, a “genetic map” is the ordered list of loci known for aparticular genome.

As used herein, “phenotype” means the detectable characteristics of acell or organism which are a manifestation of gene expression.

As used herein, a “phenotypic marker” refers to a marker that can beused to discriminate phenotypes displayed by organisms.

As used herein, “linkage” refers to relative frequency at which types ofgametes are produced in a cross. For example, if locus A has genes “A”or “a” and locus B has genes “B” or “b” and a cross between parent Iwith AABB and parent B with aabb will produce four possible gameteswhere the genes are segregated into AB, Ab, aB and ab. The nullexpectation is that there will be independent equal segregation intoeach of the four possible genotypes, i.e. with no linkage ¼ of thegametes will of each genotype. Segregation of gametes into a genotypesdiffering from ¼ are attributed to linkage.

As used herein, “linkage disequilibrium” is defined in the context ofthe relative frequency of gamete types in a population of manyindividuals in a single generation. If the frequency of allele A is p, ais p′, B is q and b is q′, then the expected frequency (with no linkagedisequilibrium) of genotype AB is pq, Ab is pq′, aB is p′q and ab isp′q′. Any deviation from the expected frequency is called linkagedisequilibrium. Two loci are said to be “genetically linked” when theyare in linkage disequilibrium.

As used herein, “quantitative trait locus (QTL)” means a locus thatcontrols to some degree numerically representable traits that areusually continuously distributed.

As used herein, the term “transgene” means nucleic acid molecules inform of DNA, such as cDNA or genomic DNA, and RNA, such as mRNA ormicroRNA, which may be single or double stranded.

As used herein, the term “inbred” means a line that has been bred forgenetic homogeneity.

As used herein, the term “hybrid” means a progeny of mating between atleast two genetically dissimilar parents. Without limitation, examplesof mating schemes include single crosses, modified single cross, doublemodified single cross, three-way cross, modified three-way cross, anddouble cross wherein at least one parent in a modified cross is theprogeny of a cross between sister lines.

As used herein, the term “tester” means a line used in a testcross withanother line wherein the tester and the lines tested are from differentgermplasm pools. A tester may be isogenic or nonisogenic.

As used herein, “resistance allele” means the isolated nucleic acidsequence that includes the polymorphic allele associated with resistanceto the disease or condition of concern.

As used herein, the term “corn” means Zea mays or maize and includes allplant varieties that can be bred with corn, including wild maizespecies.

As used herein, the term “comprising” means “including but not limitedto”.

As used herein, an “elite line” is any line that has resulted frombreeding and selection for superior agronomic performance.

As used herein, an “inducer” is a line which when crossed with anotherline promotes the formation of haploid embryos.

As used herein, “haplotype effect estimate” means a predicted effectestimate for a haplotype reflecting association with one or morephenotypic traits, wherein the associations can be made de novo or byleveraging historical haplotype-trait association data.

As used herein, “breeding value” means a calculation based on nucleicacid sequence effect estimates and nucleic acid sequence frequencyvalues, the breeding value of a specific nucleic acid sequence relativeto other nucleic acid sequences at the same locus (i.e., haplotypewindow), or across loci (i.e., haplotype windows), can also bedetermined. In other words, the change in population mean by fixing saidnucleic acid sequence is determined. In addition, in the context ofevaluating the effect of substituting a specific region in the genome,either by introgression or a transgenic event, breeding values providethe basis for comparing specific nucleic acid sequences for substitutioneffects. Also, in hybrid crops, the breeding value of nucleic acidsequences can be calculated in the context of the nucleic acid sequencein the tester used to produce the hybrid.

To the extent to which any of the preceding definitions is inconsistentwith definitions provided in any patent or non-patent referenceincorporated herein or in any reference found elsewhere, it isunderstood that the preceding definition will be used herein.

Methods and Compositions for Gray Leaf Spot Resistance in Corn

The present invention provides a method of using haploid plants toidentify genotypes associated with phenotypes of interest wherein thehaploid plant is assayed with at least one marker and associating the atleast one marker with at least one phenotypic trait. The genotype ofinterest can then be used to make decisions in a plant breeding program.Such decisions include, but are not limited to, selecting among newbreeding populations which population has the highest frequency offavorable nucleic acid sequences based on historical genotype andagronomic trait associations, selecting favorable nucleic acid sequencesamong progeny in breeding populations, selecting among parental linesbased on prediction of progeny performance, and advancing lines ingermplasm improvement activities based on presence of favorable nucleicacid sequences. Non-limiting examples of germplasm improvementactivities include line development, hybrid development, transgenicevent selection, making breeding crosses, testing and advancing a plantthrough self fertilization, using plants for transformation, usingplants for candidates for expression constructs, and using plants formutagenesis.

Non-limiting examples of breeding decisions include progeny selection,parent selection, and recurrent selection for at least one haplotype. Inanother aspect, breeding decisions relating to development of plants forcommercial release comprise advancing plants for testing, advancingplants for purity, purification of sublines during development, inbreddevelopment, variety development, and hybrid development. In yet otheraspects, breeding decisions and germplasm improvement activitiescomprise transgenic event selection, making breeding crosses, testingand advancing a plant through self-fertilization, using plants fortransformation, using plants for candidates for expression constructs,and using plants for mutagenesis.

In still another embodiment, the present invention acknowledges thatpreferred haplotypes and QTL identified by the methods presented hereinmay be advanced as candidate genes for inclusion in expressionconstructs, i.e., transgenes. Nucleic acids underlying haplotypes or QTLof interest may be expressed in plant cells by operably linking them toa promoter functional in plants. In another aspect, nucleic acidsunderlying haplotypes or QTL of interest may have their expressionmodified by double-stranded RNA-mediated gene suppression, also known asRNA interference (“RNAi”), which includes suppression mediated by smallinterfering RNAs (“siRNA”), trans-acting small interfering RNAs(“ta-siRNA”), or microRNAs (“miRNA”). Examples of RNAi methodologysuitable for use in plants are described in detail in U.S. PatentApplication Publications 2006/0200878 and 2007/0011775.

Methods are known in the art for assembling and introducing constructsinto a cell in such a manner that the nucleic acid molecule for a traitis transcribed into a functional mRNA molecule that is translated andexpressed as a protein product. For the practice of the presentinvention, conventional compositions and methods for preparing and usingconstructs and host cells are well known to one skilled in the art, seefor example, Molecular Cloning: A Laboratory Manual, 3rd Edition Volumes1, 2, and 3 (2000) J. F. Sambrook, D. W. Russell, and N. Irwin, ColdSpring Harbor Laboratory Press. Methods for making transformationconstructs particularly suited to plant transformation include, withoutlimitation, those described in U.S. Pat. Nos. 4,971,908, 4,940,835,4,769,061 and 4,757,011, all of which are herein incorporated byreference in their entirety. Transformation methods for the introductionof expression units into plants are known in the art and includeelectroporation as illustrated in U.S. Pat. No. 5,384,253;microprojectile bombardment as illustrated in U.S. Pat. Nos. 5,015,580;5,550,318; 5,538,880; 6,160,208; 6,399,861; and 6,403,865; protoplasttransformation as illustrated in U.S. Pat. No. 5,508,184; andAgrobacterium-mediated transformation as illustrated in U.S. Pat. Nos.5,635,055; 5,824,877; 5,591,616; 5,981,840; and 6,384,301.

Gray Leaf Spot Resistance

The present invention provides GLS resistance loci that are located inpublic bins in the maize genome that were not previously associated withGLS resistance.

The present invention provides 160 GLS resistance loci that are locatedin public bins in the maize genome that were not previously associatedwith GLS resistance. QTL were assigned by dividing maize chromosomalregions into 10 cM windows. A total of 178 QTL associated with GLS wereidentified, of which 158 have not been previously reported. SNP markersare also provided for monitoring the introgression of the 178 GLSresistance QTL.

In the present invention, GLS resistant loci 1-9, 14-33, 35, 38-42,44-52, 54-61, 63-71, 73-79, 81-92, 95-96, 99-106, 108-117, and 119-178have not been previously associated with GLS and are provided. SNPmarkers are also provided for monitoring the introgression of GLSresistance. In the present invention, GLS resistance loci 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, and 177 are located on chromosome 1. SNP markers used to monitorthe introgression of GLS resistance locus 1 include those selected fromthe group consisting of SEQ ID NOs: 1 through 9. SNP markers used tomonitor the introgression of GLS resistance locus 2 include thoseselected from the group consisting of SEQ ID NOs: 10 through 14. SNPmarkers used to monitor the introgression of GLS resistance locus 3include those selected from the group consisting of SEQ ID NOs: 15through 22. SNP markers used to monitor the introgression of GLSresistance locus 4 include those selected from the group consisting ofSEQ ID NOs: 23 through 30. SNP markers used to monitor the introgressionof GLS resistance locus 5 include those selected from the groupconsisting of SEQ ID NOs: 31 through 37. SNP markers used to monitor theintrogression of GLS resistance locus 6 include those selected from thegroup consisting of SEQ ID NOs: 38 through 48. SNP markers used tomonitor the introgression of GLS resistance locus 7 include thoseselected from the group consisting of SEQ ID NOs: 49 through 58. SNPmarkers used to monitor the introgression of GLS resistance locus 8include those selected from the group consisting of SEQ ID NOs: 59through 73. SNP markers used to monitor the introgression of GLSresistance locus 9 include those selected from the group consisting ofSEQ ID NOs: 74 through 86. SNP markers used to monitor the introgressionof GLS resistance locus 10 include those selected from the groupconsisting of SEQ ID NOs: 87 through 93. SNP markers used to monitor theintrogression of GLS resistance locus 11 include those selected from thegroup consisting of SEQ ID NOs: 94 through 115. SNP markers used tomonitor the introgression of GLS resistance locus 12 include thoseselected from the group consisting of SEQ ID NOs: 116 through 126. SNPmarkers used to monitor the introgression of GLS resistance locus 13include those selected from the group consisting of SEQ ID NOs: 127through 135. SNP markers used to monitor the introgression of GLSresistance locus 14 include those selected from the group consisting ofSEQ ID NOs: 136 through 139. SNP markers used to monitor theintrogression of GLS resistance locus 15 include those selected from thegroup consisting of SEQ ID NOs: 140 through 144. SNP markers used tomonitor the introgression of GLS resistance locus 16 include thoseselected from the group consisting of SEQ ID NOs: 145 through 151. SNPmarkers used to monitor the introgression of GLS resistance locus 17include those selected from the group consisting of SEQ ID NOs: 152through 162. SNP markers used to monitor the introgression of GLSresistance locus 18 include those selected from the group consisting ofSEQ ID NOs: 163 through 172. SNP markers used to monitor theintrogression of GLS resistance locus 19 include those selected from thegroup consisting of SEQ ID NOs: 173 through 178. SNP markers used tomonitor the introgression of GLS resistance locus 20 include thoseselected from the group consisting of SEQ ID NOs: 179 through 183. SNPmarkers used to monitor the introgression of GLS resistance locus 20include those selected from the group consisting of SEQ ID NOs: 179through 183. SNP markers used to monitor the introgression of GLSresistance locus 21 include those selected from the group consisting ofSEQ ID NOs: 184 through 197. SNP markers used to monitor theintrogression of GLS resistance locus 22 include those selected from thegroup consisting of SEQ ID NOs: 198 through 199. SNP markers used tomonitor the introgression of GLS resistance locus 23 include thoseselected from the group consisting of SEQ ID NOs: 200 through 201. SNPmarkers used to monitor the introgression of GLS resistance locus 24include those selected from the group consisting of SEQ ID NOs: 202through 206. SNP markers used to monitor the introgression of GLSresistance locus 25 include those selected from the group consisting ofSEQ ID NOs: 207 through 208. SNP markers used to monitor theintrogression of GLS resistance locus 26 include those selected from thegroup consisting of SEQ ID NOs: 209 through 211. SNP markers used tomonitor the introgression of GLS resistance locus 177 include SEQ ID NO:1228.

In the present invention GLS resistant loci 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, and 178 are locatedon Chromosome 2. SNP markers used to monitor the introgression of GLSresistance locus 27 include those selected from the group consisting ofSEQ ID NOs: 212 through 215. SNP markers used to monitor theintrogression of GLS resistance locus 28 include those selected from thegroup consisting of SEQ ID NOs: 216 through 221 and 1229. SNP markersused to monitor the introgression of GLS resistance locus 29 includethose selected from the group consisting of SEQ ID NOs: 222 through 224.SNP markers used to monitor the introgression of GLS resistance locus 30include those selected from the group consisting of SEQ ID NOs: 225through 231. SNP markers used to monitor the introgression of GLSresistance locus 31 include those selected from the group consisting ofSEQ ID NOs: 232 through 236. SNP markers used to monitor theintrogression of GLS resistance locus 32 include those selected from thegroup consisting of SEQ ID NOs: 237 through 242. SNP markers used tomonitor the introgression of GLS resistance locus 33 include thoseselected from the group consisting of SEQ ID NOs: 244 through 248. SNPmarkers used to monitor the introgression of GLS resistance locus 34include those selected from the group consisting of SEQ ID NOs: 249through 260. SNP markers used to monitor the introgression of GLSresistance locus 35 include those selected from the group consisting ofSEQ ID NOs: 261 through 269. SNP markers used to monitor theintrogression of GLS resistance locus 36 include those selected from thegroup consisting of SEQ ID NOs: 270 through 291. SNP markers used tomonitor the introgression of GLS resistance locus 37 include thoseselected from the group consisting of SEQ ID NOs: 292 through 303. SNPmarkers used to monitor the introgression of GLS resistance locus 38include those selected from the group consisting of SEQ ID NOs: 304through 311. SNP markers used to monitor the introgression of GLSresistance locus 39 include those selected from the group consisting ofSEQ ID NOs: 312 through 321. SNP markers used to monitor theintrogression of GLS resistance locus 40 include those selected from thegroup consisting of SEQ ID NOs: 322 through 330. SNP markers used tomonitor the introgression of GLS resistance locus 41 include thoseselected from the group consisting of SEQ ID NOs: 331 through 335. SNPmarkers used to monitor the introgression of GLS resistance locus 42include those selected from the group consisting of SEQ ID NOs: 336through 341. SNP markers used to monitor the introgression of GLSresistance locus 43 include those selected from the group consisting ofSEQ ID NOs: 342 through 348. SNP markers used to monitor theintrogression of GLS resistance locus 44 include those selected from thegroup consisting of SEQ ID NOs: 349 through 351. SNP markers used tomonitor the introgression of GLS resistance locus 45 include thoseselected from the group consisting of SEQ ID NOs: 352 through 355. SNPmarkers used to monitor the introgression of GLS resistance locus 46include those selected from the group consisting of SEQ ID NOs: 356through 360. SNP markers used to monitor the introgression of GLSresistance locus 178 include SEQ ID NO: 1229.

In the present invention GLS resistant loci 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, and 67 are locatedon Chromosome 3. SNP markers used to monitor the introgression of GLSresistance locus 47 include those selected from the group consisting ofSEQ ID NOs: 361 through 364. SNP markers used to monitor theintrogression of GLS resistance locus 48 include those selected from thegroup consisting of SEQ ID NOs: 365. SNP markers used to monitor theintrogression of GLS resistance locus 49 include those selected from thegroup consisting of SEQ ID NOs: 366. SNP markers used to monitor theintrogression of GLS resistance locus 50 include those selected from thegroup consisting of SEQ ID NOs: 367 through 369. SNP markers used tomonitor the introgression of GLS resistance locus 51 include thoseselected from the group consisting of SEQ ID NOs: 370 through 371. SNPmarkers used to monitor the introgression of GLS resistance locus 52include those selected from the group consisting of SEQ ID NOs: 372through 374. SNP markers used to monitor the introgression of GLSresistance locus 53 include those selected from the group consisting ofSEQ ID NOs: 375. SNP markers used to monitor the introgression of GLSresistance locus 54 include those selected from the group consisting ofSEQ ID NOs: 376 through 395. SNP markers used to monitor theintrogression of GLS resistance locus 55 include those selected from thegroup consisting of SEQ ID NOs: 396 through 408. SNP markers used tomonitor the introgression of GLS resistance locus 56 include thoseselected from the group consisting of SEQ ID NOs: 409 through 418. SNPmarkers used to monitor the introgression of GLS resistance locus 57include those selected from the group consisting of SEQ ID NOs: 419through 425. SNP markers used to monitor the introgression of GLSresistance locus 58 include those selected from the group consisting ofSEQ ID NOs: 426 through 433. SNP markers used to monitor theintrogression of GLS resistance locus 59 include those selected from thegroup consisting of SEQ ID NOs: 434 through 435. SNP markers used tomonitor the introgression of GLS resistance locus 60 include thoseselected from the group consisting of SEQ ID NOs: 436 through 449. SNPmarkers used to monitor the introgression of GLS resistance locus 61include those selected from the group consisting of SEQ ID NOs: 450through 458. SNP markers used to monitor the introgression of GLSresistance locus 62 include those selected from the group consisting ofSEQ ID NOs: 459 through 464. SNP markers used to monitor theintrogression of GLS resistance locus 63 include those selected from thegroup consisting of SEQ ID NOs: 465 through 471. SNP markers used tomonitor the introgression of GLS resistance locus 64 include thoseselected from the group consisting of SEQ ID NOs: 472 through 482. SNPmarkers used to monitor the introgression of GLS resistance locus 65include those selected from the group consisting of SEQ ID NOs: 483through 486. SNP markers used to monitor the introgression of GLSresistance locus 66 include those selected from the group consisting ofSEQ ID NOs: 487 through 490. SNP markers used to monitor theintrogression of GLS resistance locus 67 include those selected from thegroup consisting of SEQ ID NOs: 491 through 495.

In the present invention GLS resistant loci 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, and 87 are located onChromosome 4. SNP markers used to monitor the introgression of GLSresistance locus 68 include those selected from the group consisting ofSEQ ID NOs: 496 through 499. SNP markers used to monitor theintrogression of GLS resistance locus 69 include those selected from thegroup consisting of SEQ ID NOs: 500 through 502. SNP markers used tomonitor the introgression of GLS resistance locus 70 include thoseselected from the group consisting of SEQ ID NOs: 503 through 504. SNPmarkers used to monitor the introgression of GLS resistance locus 71include those selected from the group consisting of SEQ ID NOs: 505through 507. SNP markers used to monitor the introgression of GLSresistance locus 72 include those selected from the group consisting ofSEQ ID NOs: 508 through 511. SNP markers used to monitor theintrogression of GLS resistance locus 73 include those selected from thegroup consisting of SEQ ID NOs: 512 through 515. SNP markers used tomonitor the introgression of GLS resistance locus 74 include thoseselected from the group consisting of SEQ ID NOs: 516 through 530. SNPmarkers used to monitor the introgression of GLS resistance locus 75include those selected from the group consisting of SEQ ID NOs: 531through 551. SNP markers used to monitor the introgression of GLSresistance locus 76 include those selected from the group consisting ofSEQ ID NOs: 552 through 567. SNP markers used to monitor theintrogression of GLS resistance locus 77 include those selected from thegroup consisting of SEQ ID NOs: 568 through 578. SNP markers used tomonitor the introgression of GLS resistance locus 78 include thoseselected from the group consisting of SEQ ID NOs: 579 through 586. SNPmarkers used to monitor the introgression of GLS resistance locus 79include those selected from the group consisting of SEQ ID NOs: 587through 590. SNP markers used to monitor the introgression of GLSresistance locus 80 include those selected from the group consisting ofSEQ ID NOs: 591 through 603. SNP markers used to monitor theintrogression of GLS resistance locus 81 include those selected from thegroup consisting of SEQ ID NOs: 604 through 617. SNP markers used tomonitor the introgression of GLS resistance locus 82 include thoseselected from the group consisting of SEQ ID NOs: 618 through 625. SNPmarkers used to monitor the introgression of GLS resistance locus 83include those selected from the group consisting of SEQ ID NOs: 626through 632. SNP markers used to monitor the introgression of GLSresistance locus 84 include those selected from the group consisting ofSEQ ID NOs: 633 through 639. SNP markers used to monitor theintrogression of GLS resistance locus 85 include those selected from thegroup consisting of SEQ ID NOs: 640 through 644. SNP markers used tomonitor the introgression of GLS resistance locus 86 include thoseselected from the group consisting of SEQ ID NOs: 645 through 653.

SNP markers used to monitor the introgression of GLS resistance locus 87include those selected from the group consisting of SEQ ID NOs: 654through 656.

In the present invention GLS resistant loci 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 101, 102, 103, and 104 are located onChromosome 5. SNP markers used to monitor the introgression of GLSresistance locus 88 include those selected from the group consisting ofSEQ ID NOs: 657 through 660. SNP markers used to monitor theintrogression of GLS resistance locus 89 include those selected from thegroup consisting of SEQ ID NOs: 661 through 668. SNP markers used tomonitor the introgression of GLS resistance locus 90 include thoseselected from the group consisting of SEQ ID NOs: 669 through 670. SNPmarkers used to monitor the introgression of GLS resistance locus 91include those selected from the group consisting of SEQ ID NOs: 671through 674. SNP markers used to monitor the introgression of GLSresistance locus 92 include those selected from the group consisting ofSEQ ID NOs: 675 through 678. SNP markers used to monitor theintrogression of GLS resistance locus 93 include those selected from thegroup consisting of SEQ ID NOs: 679 through 692. SNP markers used tomonitor the introgression of GLS resistance locus 94 include thoseselected from the group consisting of SEQ ID NOs: 693 through 709. SNPmarkers used to monitor the introgression of GLS resistance locus 95include those selected from the group consisting of SEQ ID NOs: 710through 721. SNP markers used to monitor the introgression of GLSresistance locus 96 include those selected from the group consisting ofSEQ ID NOs: 722 through 730. SNP markers used to monitor theintrogression of GLS resistance locus 97 include those selected from thegroup consisting of SEQ ID NOs: 731 through 738. SNP markers used tomonitor the introgression of GLS resistance locus 98 include thoseselected from the group consisting of SEQ ID NOs: 739 through 740. SNPmarkers used to monitor the introgression of GLS resistance locus 99include those selected from the group consisting of SEQ ID NOs: 741through 748. SNP markers used to monitor the introgression of GLSresistance locus 100 include those selected from the group consisting ofSEQ ID NOs: 749 through 754. SNP markers used to monitor theintrogression of GLS resistance locus 101 include those selected fromthe group consisting of SEQ ID NOs: 755 through 760. SNP markers used tomonitor the introgression of GLS resistance locus 102 include thoseselected from the group consisting of SEQ ID NOs: 761 through 762. SNPmarkers used to monitor the introgression of GLS resistance locus 103include those selected from the group consisting of SEQ ID NOs: 763through 771. SNP markers used to monitor the introgression of GLSresistance locus 104 include those selected from the group consisting ofSEQ ID NOs: 772 through 776.

In the present invention GLS resistant loci 105, 106, 107, 108, 109,110, 111, 112, 113, 114, 115, 116, and 117 are located on Chromosome 6.SNP markers used to monitor the introgression of GLS resistance locus105 include those selected from the group consisting of SEQ ID NOs: 777through 780. SNP markers used to monitor the introgression of GLSresistance locus 106 include those selected from the group consisting ofSEQ ID NOs: 781 through 812. SNP markers used to monitor theintrogression of GLS resistance locus 107 include those selected fromthe group consisting of SEQ ID NOs: 813 through 820. SNP markers used tomonitor the introgression of GLS resistance locus 108 include thoseselected from the group consisting of SEQ ID NOs: 821 through 829 and1232. SNP markers used to monitor the introgression of GLS resistancelocus 109 include those selected from the group consisting of SEQ IDNOs: 830 through 834. SNP markers used to monitor the introgression ofGLS resistance locus 110 include those selected from the groupconsisting of SEQ ID NOs: 835 through 845 and 1231. SNP markers used tomonitor the introgression of GLS resistance locus 111 include thoseselected from the group consisting of SEQ ID NOs: 846 through 854. SNPmarkers used to monitor the introgression of GLS resistance locus 112include those selected from the group consisting of SEQ ID NOs: 855through 863. SNP markers used to monitor the introgression of GLSresistance locus 113 include those selected from the group consisting ofSEQ ID NOs: 864 through 869. SNP markers used to monitor theintrogression of GLS resistance locus 114 include those selected fromthe group consisting of SEQ ID NOs: 870 through 873. SNP markers used tomonitor the introgression of GLS resistance locus 115 include thoseselected from the group consisting of SEQ ID NOs: 874 through 875. SNPmarkers used to monitor the introgression of GLS resistance locus 116include those selected from the group consisting of SEQ ID NOs: 876through 883. SNP markers used to monitor the introgression of GLSresistance locus 117 include those selected from the group consisting ofSEQ ID NOs: 884 through 889 and 1360.

In the present invention GLS resistant loci 118, 119, 120, 121, 122,123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, and 135 arelocated on Chromosome 7. SNP markers used to monitor the introgressionof GLS resistance locus 118 include those selected from the groupconsisting of SEQ ID NOs: 890 through 891. SNP markers used to monitorthe introgression of GLS resistance locus 119 include those selectedfrom the group consisting of SEQ ID NOs: 892. SNP markers used tomonitor the introgression of GLS resistance locus 120 include thoseselected from the group consisting of SEQ ID NOs: 893. SNP markers usedto monitor the introgression of GLS resistance locus 121 include thoseselected from the group consisting of SEQ ID NOs: 894. SNP markers usedto monitor the introgression of GLS resistance locus 122 include thoseselected from the group consisting of SEQ ID NOs: 895 through 898. SNPmarkers used to monitor the introgression of GLS resistance locus 123include those selected from the group consisting of SEQ ID NOs: 899through 907. SNP markers used to monitor the introgression of GLSresistance locus 124 include those selected from the group consisting ofSEQ ID NOs: 908 through 932. SNP markers used to monitor theintrogression of GLS resistance locus 125 include those selected fromthe group consisting of SEQ ID NOs: 933 through 939. SNP markers used tomonitor the introgression of GLS resistance locus 126 include thoseselected from the group consisting of SEQ ID NOs: 940 through 943. SNPmarkers used to monitor the introgression of GLS resistance locus 127include those selected from the group consisting of SEQ ID NOs: 944through 953 and 1233. SNP markers used to monitor the introgression ofGLS resistance locus 128 include those selected from the groupconsisting of SEQ ID NOs: 954 through 963. SNP markers used to monitorthe introgression of GLS resistance locus 129 include those selectedfrom the group consisting of SEQ ID NOs: 964 through 968. SNP markersused to monitor the introgression of GLS resistance locus 130 includethose selected from the group consisting of SEQ ID NOs: 969 through 971.SNP markers used to monitor the introgression of GLS resistance locus131 include those selected from the group consisting of SEQ ID NOs: 972through 976. SNP markers used to monitor the introgression of GLSresistance locus 132 include those selected from the group consisting ofSEQ ID NOs: 977. SNP markers used to monitor the introgression of GLSresistance locus 133 include those selected from the group consisting ofSEQ ID NOs: 978 through 982. SNP markers used to monitor theintrogression of GLS resistance locus 134 include those selected fromthe group consisting of SEQ ID NOs: 983 through 990. SNP markers used tomonitor the introgression of GLS resistance locus 135 include thoseselected from the group consisting of SEQ ID NOs: 991 through 996.

In the present invention GLS resistant loci 136, 137, 138, 139, 140,141, 142, 143, 144, 145, 146, 147, 148, and 149 are located onChromosome 8. SNP markers used to monitor the introgression of GLSresistance locus 136 include those selected from the group consisting ofSEQ ID NOs: 997 through 1000. SNP markers used to monitor theintrogression of GLS resistance locus 137 include those selected fromthe group consisting of SEQ ID NOs: 1001 through 1003. SNP markers usedto monitor the introgression of GLS resistance locus 138 include thoseselected from the group consisting of SEQ ID NOs: 1004 through 1010. SNPmarkers used to monitor the introgression of GLS resistance locus 139include those selected from the group consisting of SEQ ID NOs: 1011through 1015. SNP markers used to monitor the introgression of GLSresistance locus 140 include those selected from the group consisting ofSEQ ID NOs: 1016 through 1022. SNP markers used to monitor theintrogression of GLS resistance locus 141 include those selected fromthe group consisting of SEQ ID NOs: 1023 through 1031. SNP markers usedto monitor the introgression of GLS resistance locus 142 include thoseselected from the group consisting of SEQ ID NOs: 1032 through 1046. SNPmarkers used to monitor the introgression of GLS resistance locus 143include those selected from the group consisting of SEQ ID NOs: 1047through 1050. SNP markers used to monitor the introgression of GLSresistance locus 144 include those selected from the group consisting ofSEQ ID NOs: 1051 through 1060. SNP markers used to monitor theintrogression of GLS resistance locus 145 include those selected fromthe group consisting of SEQ ID NOs: 1061 through 1062. SNP markers usedto monitor the introgression of GLS resistance locus 146 include thoseselected from the group consisting of SEQ ID NOs: 1063 through 1069. SNPmarkers used to monitor the introgression of GLS resistance locus 147include those selected from the group consisting of SEQ ID NOs: 1070through 1072. SNP markers used to monitor the introgression of GLSresistance locus 148 include those selected from the group consisting ofSEQ ID NOs: 1073 through 1075. SNP markers used to monitor theintrogression of GLS resistance locus 149 include those selected fromthe group consisting of SEQ ID NOs: 1076 through 1078.

In the present invention GLS resistant loci 150, 151, 152, 153, 154,155, 156, 157, 158, 159, 160, 161, 162, 163, 164, and 165 are located onChromosome 9. SNP markers used to monitor the introgression of GLSresistance locus 150 include those selected from the group consisting ofSEQ ID NOs: 1079 through 1081. SNP markers used to monitor theintrogression of GLS resistance locus 151 include those selected fromthe group consisting of SEQ ID NOs: 1082 through 1086. SNP markers usedto monitor the introgression of GLS resistance locus 152 include thoseselected from the group consisting of SEQ ID NOs: 1087. SNP markers usedto monitor the introgression of GLS resistance locus 153 include thoseselected from the group consisting of SEQ ID NOs: 1088 through 1091. SNPmarkers used to monitor the introgression of GLS resistance locus 154include those selected from the group consisting of SEQ ID NOs: 1092through 1096. SNP markers used to monitor the introgression of GLSresistance locus 155 include those selected from the group consisting ofSEQ ID NOs: 1097 through 1098. SNP markers used to monitor theintrogression of GLS resistance locus 156 include those selected fromthe group consisting of SEQ ID NOs: 1099 through 1110. SNP markers usedto monitor the introgression of GLS resistance locus 157 include thoseselected from the group consisting of SEQ ID NOs: 1111 through 1118. SNPmarkers used to monitor the introgression of GLS resistance locus 158include those selected from the group consisting of SEQ ID NOs: 1119through 1133 and 1127. SNP markers used to monitor the introgression ofGLS resistance locus 159 include those selected from the groupconsisting of SEQ ID NOs: 1134 through 1142. SNP markers used to monitorthe introgression of GLS resistance locus 160 include those selectedfrom the group consisting of SEQ ID NOs: 1143 through 1150. SNP markersused to monitor the introgression of GLS resistance locus 161 includethose selected from the group consisting of SEQ ID NOs: 1151 through1157. SNP markers used to monitor the introgression of GLS resistancelocus 162 include those selected from the group consisting of SEQ IDNOs: 1158 through 1159. SNP markers used to monitor the introgression ofGLS resistance locus 163 include those selected from the groupconsisting of SEQ ID NOs: 1160 through 1164. SNP markers used to monitorthe introgression of GLS resistance locus 164 include those selectedfrom the group consisting of SEQ ID NOs: 1165. SNP markers used tomonitor the introgression of GLS resistance locus 165 include thoseselected from the group consisting of SEQ ID NOs: 1166 through 1167.

In the present invention GLS resistant loci 166, 167, 168, 169, 170,171, 172, 173, 174, 175, and 176 are located on Chromosome 10. SNPmarkers used to monitor the introgression of GLS resistance locus 166include those selected from the group consisting of SEQ ID NOs: 1168.SNP markers used to monitor the introgression of GLS resistance locus167 include those selected from the group consisting of SEQ ID NOs: 1169through 1172. SNP markers used to monitor the introgression of GLSresistance locus 168 include those selected from the group consisting ofSEQ ID NOs: 1173 through 1177. SNP markers used to monitor theintrogression of GLS resistance locus 169 include those selected fromthe group consisting of SEQ ID NOs: 1178 through 1192. SNP markers usedto monitor the introgression of GLS resistance locus 170 include thoseselected from the group consisting of SEQ ID NOs: 1193 through 1203 and1361. SNP markers used to monitor the introgression of GLS resistancelocus 171 include those selected from the group consisting of SEQ IDNOs: 1204 through 1210. SNP markers used to monitor the introgression ofGLS resistance locus 172 include those selected from the groupconsisting of SEQ ID NOs: 1211 through 1215. SNP markers used to monitorthe introgression of GLS resistance locus 173 include those selectedfrom the group consisting of SEQ ID NOs: 1216 through 1219. SNP markersused to monitor the introgression of GLS resistance locus 174 includethose selected from the group consisting of SEQ ID NOs: 1220 through1221. SNP markers used to monitor the introgression of GLS resistancelocus 175 include those selected from the group consisting of SEQ IDNOs: 1222 through 1226. SNP markers used to monitor the introgression ofGLS resistance locus 176 include SEQ ID NO: 1227.

Exemplary marker assays for screening for GLS resistance loci areprovided in Tables 3, 4, and 5. Illustrative GLS resistance locus 173SNP marker DNA sequence SEQ ID NO: 1219 can be amplified using theprimers indicated as SEQ ID NOs: 1304 through 1305 and detected withprobes indicated as SEQ ID NOs: 1306 through 1307. Illustrative GLSresistance locus 57 SNP marker DNA sequence SEQ ID NO: 421 can beamplified using the primers indicated as SEQ ID NOs: 1308 through 1309and detected with probes indicated as SEQ ID NOs: 1310 through 1311.Illustrative GLS resistance locus 64 SNP marker DNA sequence SEQ ID NO:481 can be amplified using the primers indicated as SEQ ID NOs: 1312through 1313 and detected with probes indicated as SEQ ID NOs: 1314through 1315. Illustrative GLS resistance locus 176 SNP marker DNAsequence SEQ ID NO: 1127 can be amplified using the primers indicated asSEQ ID NOs: 1316 through 1317 and detected with probes indicated as SEQID NOs: 1318 through 1319. Illustrative oligonucleotide hybridizationprobes for GLS resistance locus 173 SNP marker DNA sequence SEQ ID NO:1219 are provided as SEQ ID NO: 1320 and SEQ ID NO 1321. Illustrativeoligonucleotide hybridization probes for GLS resistance locus 57 SNPmarker DNA sequence SEQ ID NO: 421 are provided as SEQ ID NO: 1322 andSEQ ID NO: 1323. Illustrative oligonucleotide hybridization probes forGLS resistance locus 64 SNP marker DNA sequence SEQ ID NO: 481 areprovided as SEQ ID NO: 1324 and SEQ ID NO: 1325. Illustrativeoligonucleotide hybridization probes for GLS resistance locus 176 SNPmarker DNA sequence SEQ ID NO: 1127 are provided as SEQ ID NO: 1326 andSEQ ID NO: 1327. An illustrative probe for single base extension assaysfor GLS resistance locus 173 SNP marker DNA sequence SEQ ID NO: 1219 isprovided as SEQ ID NO: 1328. An illustrative probe for single baseextension assays for GLS resistance locus 57 SNP marker DNA sequence SEQID NO: 421 is provided as SEQ ID NO: 1329. An illustrative probe forsingle base extension assays for GLS resistance locus 64 SNP marker DNAsequence SEQ ID NO: 481 is provided as SEQ ID NO: 1330. An illustrativeprobe for single base extension assays for GLS resistance locus 176 SNPmarker DNA sequence SEQ ID NO: 1127 is provided as SEQ ID NO: 1331.

The present invention also provides a corn plant comprising a nucleicacid molecule selected from the group consisting of SEQ ID NO: 1 through1233, 1360, and 1361, fragments thereof, and complements of both.

As used herein, GLS refers to any Gray Leaf Spot variant or isolate. Acorn plant of the present invention can be resistant to one or morefungi capable of causing or inducing GLS. In one aspect, the presentinvention provides plants resistant to GLS as well as methods andcompositions for screening corn plants for resistance or susceptibilityto GLS, caused by the genus Cercospora. In a preferred aspect, thepresent invention provides methods and compositions for screening cornplants for resistance or susceptibility to C. zea-maydis. In anotheraspect, the present invention provides plants resistant to and methodsand compositions for screening corn plants for resistance orsusceptibility to C. zea-maydis strain “Type I.” In a further aspect,the present invention provides plants resistant to and methods andcompositions for screening corn plants for resistance or susceptibilityto C. zea-maydis strain “Type II.” In an additional aspect, the presentinvention provides plants resistant to and methods and compositions forscreening corn plants for resistance or susceptibility to C. sorghi var.maydis.

In an aspect, the plant is selected from the genus Zea. In anotheraspect, the plant is selected from the species Zea mays. In a furtheraspect, the plant is selected from the subspecies Zea mays L. ssp. mays.In an additional aspect, the plant is selected from the group Zea maysL. subsp. mays Indentata, otherwise known as dent corn. In anotheraspect, the plant is selected from the group Zea mays L. subsp. maysIndurata, otherwise known as flint corn. In another an aspect, the plantis selected from the group Zea mays L. subsp. mays Saccharata, otherwiseknown as sweet corn. In another aspect, the plant is selected from thegroup Zea mays L. subsp. mays Amylacea, otherwise known as flour corn.In a further aspect, the plant is selected from the group Zea mays L.subsp. mays Everta, otherwise known as pop corn. Zea plants includehybrids, inbreds, partial inbreds, or members of defined or undefinedpopulations.

Plants of the present invention can be a corn plant that is veryresistant, resistant, substantially resistant, mid-resistant,comparatively resistant, partially resistant, mid-susceptible, orsusceptible.

In a preferred aspect, the present invention provides a corn plant to beassayed for resistance or susceptibility to GLS by any method todetermine whether a corn plant is very resistant, resistant,substantially resistant, mid-resistant, comparatively resistant,partially resistant, mid-susceptible, or susceptible. Phenotyping forGLS is based on visually screening plants to determine percentage ofinfected leaf area. The percentage of leaf area infected is used to rateplants on a scale of 1 (very resistant) to 9 (susceptible). Diseaseresistance is evaluated visually after pollination. The infection can benatural or from artificial inoculation.

A disease resistance QTL of the present invention may be introduced intoan elite corn inbred line.

In another aspect, the corn plant can show a comparative resistancecompared to a non-resistant control corn plant. In this aspect, acontrol corn plant will preferably be genetically similar except for theGLS resistant allele or alleles in question. Such plants can be grownunder similar conditions with equivalent or near equivalent exposure tothe pathogen. In this aspect, the resistant plant or plants has lessthan 25%, 15%, 10%, 5%, 2% or 1% of leaf area infected.

A disease resistance QTL of the present invention may be introduced intoan elite corn inbred line. An “elite line” is any line that has resultedfrom breeding and selection for superior agronomic performance.

A GLS resistance QTL of the present invention may also be introducedinto an elite corn plant comprising one or more transgenes conferringherbicide tolerance, increased yield, insect control, fungal diseaseresistance, virus resistance, nematode resistance, bacterial diseaseresistance, mycoplasma disease resistance, modified oils production,high oil production, high protein production, germination and seedlinggrowth control, enhanced animal and human nutrition, low raffinose,environmental stress resistant, increased digestibility, industrialenzymes, pharmaceutical proteins, peptides and small molecules, improvedprocessing traits, improved flavor, nitrogen fixation, hybrid seedproduction, reduced allergenicity, biopolymers, and biofuels amongothers. In one aspect, the herbicide tolerance is selected from thegroup consisting of glyphosate, dicamba, glufosinate, sulfonylurea,bromoxynil and norflurazon herbicides. These traits can be provided bymethods of plant biotechnology as transgenes in corn.

A disease resistant QTL allele or alleles can be introduced from anyplant that contains that allele (donor) to any recipient corn plant. Inone aspect, the recipient corn plant can contain additional GLSresistant loci. In another aspect, the recipient corn plant can containa transgene. In another aspect, while maintaining the introduced QTL,the genetic contribution of the plant providing the disease resistantQTL can be reduced by back-crossing or other suitable approaches. In oneaspect, the nuclear genetic material derived from the donor material inthe corn plant can be less than or about 50%, less than or about 25%,less than or about 13%, less than or about 5%, 3%, 2% or 1%, but thatgenetic material contains the GLS resistant locus or loci of interest.

It is further understood that a corn plant of the present invention mayexhibit the characteristics of any relative maturity group. In anaspect, the maturity group is selected from the group consisting ofRM90-95, RM 95-100, RM 100-105, RM 105-110, RM 110-115, and RM 115-120.

An allele of a QTL can, of course, comprise multiple genes or othergenetic factors even within a contiguous genomic region or linkagegroup, such as a haplotype. As used herein, an allele of a diseaseresistance locus can therefore encompass more than one gene or othergenetic factor where each individual gene or genetic component is alsocapable of exhibiting allelic variation and where each gene or geneticfactor is also capable of eliciting a phenotypic effect on thequantitative trait in question. In an aspect of the present inventionthe allele of a QTL comprises one or more genes or other genetic factorsthat are also capable of exhibiting allelic variation. The use of theterm “an allele of a QTL” is thus not intended to exclude a QTL thatcomprises more than one gene or other genetic factor. Specifically, an“allele of a QTL” in the present in the invention can denote a haplotypewithin a haplotype window wherein a phenotype can be disease resistance.A haplotype window is a contiguous genomic region that can be defined,and tracked, with a set of one or more polymorphic markers wherein thepolymorphisms indicate identity by descent. A haplotype within thatwindow can be defined by the unique fingerprint of alleles at eachmarker. As used herein, an allele is one of several alternative forms ofa gene occupying a given locus on a chromosome. When all the allelespresent at a given locus on a chromosome are the same, that plant ishomozygous at that locus. If the alleles present at a given locus on achromosome differ, that plant is heterozygous at that locus. Plants ofthe present invention may be homozygous or heterozygous at anyparticular GLS locus or for a particular polymorphic marker.

The present invention also provides for parts of the plants of thepresent invention. Plant parts, without limitation, include seed,endosperm, ovule and pollen. In a particularly preferred aspect of thepresent invention, the plant part is a seed.

The present invention also provides a container of corn in which greaterthan 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the seeds comprising 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137,138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151,152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165,166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, and 178 GLSresistant loci where one or more alleles at one or more of their lociare selected from the group consisting of SEQ ID NOs 1-1233, 1360, and1361.

The container of corn seeds can contain any number, weight, or volume ofseeds. For example, a container can contain at least, or greater than,about 10, 25, 50, 100, 200, 300, 400, 500, 600, 700, 80, 90, 1000, 1500,2000, 2500, 3000, 3500, 4000 or more seeds. In another aspect, acontainer can contain about, or greater than about, 1 gram, 5 grams, 10grams, 15 grams, 20 grams, 25 grams, 50 grams, 100 grams, 250 grams, 500grams, or 1000 grams of seeds. Alternatively, the container can containat least, or greater than, about 0 ounces, 1 ounce, 5 ounces, 10 ounces,1 pound, 2 pounds, 3 pounds, 4 pounds, 5 pounds, 10 pounds, 15 pounds,20 pounds, 25 pounds, or 50 pounds or more seeds.

Containers of corn seeds can be any container available in the art. Forexample, a container can be a box, a bag, a can, a packet, a pouch, atape roll, a pail, or a tube.

In another aspect, the seeds contained in the containers of corn seedscan be treated or untreated corn seeds. In one aspect, the seeds can betreated to improve germination, for example, by priming the seeds, or bydisinfection to protect against seed-born pathogens. In another aspect,seeds can be coated with any available coating to improve, for example,plantability, seed emergence, and protection against seed-bornpathogens. Seed coating can be any form of seed coating including, butnot limited to, pelleting, film coating, and encrustments.

Plants of the present invention may also be grown in culture andregenerated. Methods for the regeneration of Zea mays plants fromvarious tissue types and methods for the tissue culture of Zea mays areknown in the art (for example, Bhaskaran et al., 1990 Crop Sci.30:1328-1336). Regeneration techniques for plants such as Zea mays canuse as the starting material a variety of tissue or cell types. With Zeamays in particular, regeneration processes have been developed thatbegin with certain differentiated tissue types such as meristems,(Sairam et al., 2003 Genome 46:323-3). Regeneration of mature Zea maysplants from tissue culture by organogenesis and embryogenesis has alsobeen reported (Wang 1987 Plant Cell. Rep. 6:360-362; Chang 1983 PlantCell. Rep. 2:18-185; Green et al. 1975 Crop Sci. 15:417-421). Recently,regeneration of corn from split seeds was also reported (Al-Abed et al.,2006 Planta 223:1355-1366).

The present invention also provides a disease resistant corn plantselected for by screening for disease resistance or susceptibility inthe corn plant, the selection comprising interrogating genomic nucleicacids for the presence of a marker molecule that is genetically linkedto an allele of a QTL associated with disease resistance in the cornplant, where the allele of a QTL is also located on a linkage groupassociated with disease resistant GLS.

Nucleic Acids

The present invention includes isolated nucleic acid molecules. Suchmolecules include those nucleic acid molecules capable of detecting apolymorphism genetically or physically linked to a Gray Leaf SpotResistance loci. In certain embodiments, the isolated nucleic acidmolecule is selected from the group consisting of SEQ ID NOs: 1-62,64-70, 72-156, 158-172, 174-187, 189-377, 379, 380, 382-409, 411-459,461-1233, 1360, 1361, fragments thereof, complements thereof, andnucleic acid molecules capable of specifically hybridizing to one ormore of these nucleic acid molecules.

In one embodiment, an isolated nucleic acid molecule of the presentinvention includes those that will specifically hybridize to one or moreof the nucleic acid molecules set forth in of SEQ ID NOs: 1-62, 64-70,72-156, 158-172, 174-187, 189-377, 379, 380, 382-409, 411-459, 461-1233,1360, 1361 and complements thereof under stringent hybridizationconditions of 20×SSC and about 65 degrees C. In a further aspect of thepresent invention, a preferred marker nucleic acid molecule of thepresent invention shares between 95% and 100% sequence identity with thesequences set forth in SEQ ID NOs: 1-62, 64-70, 72-156, 158-172,174-187, 189-377, 379, 380, 382-409, 411-459, 461-1233, 1360, 1361 orcomplements thereof or fragments of either. In a more preferred aspectof the present invention, a preferred marker nucleic acid molecule ofthe present invention shares between 98% and 100% sequence identity withthe nucleic acid sequence set forth in SEQ ID NOs: 1-62, 64-70, 72-156,158-172, 174-187, 189-377, 379, 380, 382-409, 411-459, 461-1233, 1360,1361 or complement thereof or fragments of either.

Nucleic acid molecules or fragments thereof are capable of specificallyhybridizing to other nucleic acid molecules under certain circumstances.As used herein, two nucleic acid molecules are capable of specificallyhybridizing to one another if the two molecules are capable of formingan anti-parallel, double-stranded nucleic acid structure. A nucleic acidmolecule is the “complement” of another nucleic acid molecule if theyexhibit complete complementarity. As used herein, molecules are exhibit“complete complementarity” when every nucleotide of one of the moleculesis complementary to a nucleotide of the other. Two molecules are“minimally complementary” if they can hybridize to one another withsufficient stability to permit them to remain annealed to one anotherunder at least conventional “low-stringency” conditions. Similarly, themolecules are “complementary” if they can hybridize to one another withsufficient stability to permit them to remain annealed to one anotherunder conventional “high-stringency” conditions. Conventional stringencyconditions are described by Sambrook et al., In: Molecular Cloning, ALaboratory Manual, 2nd Edition, Cold Spring Harbor Press, Cold SpringHarbor, N.Y. (1989), and by Haymes et al., In: Nucleic AcidHybridization, A Practical Approach, IRL Press, Washington, D.C. (1985).Departures from complete complementarity are therefore permissible, aslong as such departures do not completely preclude the capacity of themolecules to form a double-stranded structure. In order for a nucleicacid molecule to serve as a primer or probe it need only be sufficientlycomplementary in sequence to be able to form a stable double-strandedstructure under the particular solvent and salt concentrations employed.

As used herein, a substantially homologous sequence is a nucleic acidsequence that will specifically hybridize to the complement of thenucleic acid sequence to which it is being compared under highstringency conditions. The nucleic-acid probes and primers of thepresent invention can hybridize under stringent conditions to a targetDNA sequence. The term “stringent hybridization conditions” is definedas conditions under which a probe or primer hybridizes specifically witha target sequence(s) and not with non-target sequences, as can bedetermined empirically. The term “stringent conditions” is functionallydefined with regard to the hybridization of a nucleic-acid probe to atarget nucleic acid (i.e., to a particular nucleic-acid sequence ofinterest) by the specific hybridization procedure discussed in Sambrooket al., 1989, at 9.52-9.55. See also, Sambrook et al., 1989 at9.47-9.52, 9.56-9.58; Kanehisa 1984 Nucl. Acids Res. 12:203-213; andWetmur et al., 1968 J. Mol. Biol. 31:349-370. Appropriate stringencyconditions that promote DNA hybridization are, for example, 6.0× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by a wash of2.0×SSC at 50° C., are known to those skilled in the art or can be foundin Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.,1989, 6.3.1-6.3.6. For example, the salt concentration in the wash stepcan be selected from a low stringency of about 2.0×SSC at 50° C. to ahigh stringency of about 0.2×SSC at 50° C. In addition, the temperaturein the wash step can be increased from low stringency conditions at roomtemperature, about 22° C., to high stringency conditions at about 65° C.Both temperature and salt may be varied, or either the temperature orthe salt concentration may be held constant while the other variable ischanged.

For example, hybridization using DNA or RNA probes or primers can beperformed at 65° C. in 6×SSC, 0.5% SDS, 5×Denhardt's, 100 μg/mLnonspecific DNA (e.g., sonicated salmon sperm DNA) with washing at0.5×SSC, 0.5% SDS at 65° C., for high stringency.

It is contemplated that lower stringency hybridization conditions suchas lower hybridization and/or washing temperatures can be used toidentify related sequences having a lower degree of sequence similarityif specificity of binding of the probe or primer to target sequence(s)is preserved. Accordingly, the nucleotide sequences of the presentinvention can be used for their ability to selectively form duplexmolecules with complementary stretches of DNA, RNA, or cDNA fragments.

A fragment of a nucleic acid molecule provided herein can be of anysize. Fragments provided herein include, but are not limited to,fragments of nucleic acid sequences set forth in SEQ ID NO: 1-62, 64-70,72-156, 158-172, 174-187, 189-377, 379, 380, 382-409, 411-459, 461-1233,1360 and 1361. In one aspect, a fragment of a nucleic acid molecule canbe 15 to 25, 15 to 30, 15 to 40, 15 to 50, 15 to 100, 20 to 25, 20 to30, 20 to 40, 20 to 50, 20 to 100, 25 to 30, 25 to 40, 25 to 50, 25 to100, 30 to 40, 30 to 50, or 30 to 100 nucleotides in length. In anotheraspect, the fragment can be greater than 10, 15, 20, 25, 30, 35, 40, 50,100, or 250 nucleotides in length.

Additional genetic markers can be used to select plants with an alleleof a QTL associated with Gray Leaf Spot resistance of the presentinvention. Examples of public marker databases include, but are notlimited to, the Maize Genome Database located on the world wide web atwww.maizegdb.org, the MaizeSeq database located on the world wide web atwww.www.maizeseq.org, the Panzea maize marker and map database locatedon the world wide web at www.panzea.org, and the MAGI database locatedon the world wide web at www.plantgenomics.iastate.edu/maize.

Marker Technology

Genetic markers of the present invention include “dominant” or“codominant” markers. “Codominant markers” reveal the presence of two ormore alleles (two per diploid individual). “Dominant markers” reveal thepresence of only a single allele. The presence of the dominant markerphenotype (e.g., a band of DNA) is an indication that one allele ispresent in either the homozygous or heterozygous condition. The absenceof the dominant marker phenotype (e.g., absence of a DNA band) is merelyevidence that “some other” undefined allele is present. In the case ofpopulations where individuals are predominantly homozygous and loci arepredominantly dimorphic, dominant and codominant markers can be equallyvaluable. As populations become more heterozygous and multiallelic,codominant markers often become more informative of the genotype thandominant markers.

In another embodiment, markers, such as single sequence repeat markers(SSR), AFLP markers, RFLP markers, RAPD markers, phenotypic markers,isozyme markers, single nucleotide polymorphisms (SNPs), insertions ordeletions (Indels), single feature polymorphisms (SFPs, for example, asdescribed in Borevitz et al., 2003 Gen. Res. 13:513-523), microarraytranscription profiles, DNA-derived sequences, and RNA-derived sequencesthat are genetically linked to or correlated with alleles of a QTL ofthe present invention can be utilized.

In one embodiment, nucleic acid-based analyses for the presence orabsence of the genetic polymorphism can be used for the selection ofseeds in a breeding population. A wide variety of genetic markers forthe analysis of genetic polymorphisms are available and known to thoseof skill in the art. The analysis may be used to select for genes, QTL,alleles, or genomic regions (haplotypes) that comprise or are linked toa genetic marker.

Herein, nucleic acid analysis methods are known in the art and include,but are not limited to, PCR-based detection methods (for example, TaqManassays), microarray methods, and nucleic acid sequencing methods. In oneembodiment, the detection of polymorphic sites in a sample of DNA, RNA,or cDNA may be facilitated through the use of nucleic acid amplificationmethods. Such methods specifically increase the concentration ofpolynucleotides that span the polymorphic site, or include that site andsequences located either distal or proximal to it. Such amplifiedmolecules can be readily detected by gel electrophoresis, fluorescencedetection methods, or other means.

A method of achieving such amplification employs the polymerase chainreaction (PCR) (Mullis et al., 1986 Cold Spring Harbor Symp. Quant.Biol. 51:263-273; European Patent 50,424; European Patent 84,796;European Patent 258,017; European Patent 237,362; European Patent201,184; U.S. Pat. No. 4,683,202; U.S. Pat. No. 4,582,788; and U.S. Pat.No. 4,683,194), using primer pairs that are capable of hybridizing tothe proximal sequences that define a polymorphism in its double-strandedform.

Polymorphisms in DNA sequences can be detected or typed by a variety ofeffective methods well known in the art including, but not limited to,those disclosed in U.S. Pat. Nos. 5,468,613 and 5,217,863; 5,210,015;5,876,930; 6,030,787; 6,004,744; 6,013,431; 5,595,890; 5,762,876;5,945,283; 5,468,613; 6,090,558; 5,800,944; and 5,616,464, all of whichare incorporated herein by reference in their entireties. However, thecompositions and methods of this invention can be used in conjunctionwith any polymorphism typing method to type polymorphisms in corngenomic DNA samples. These corn genomic DNA samples used include but arenot limited to, corn genomic DNA isolated directly from a corn plant,cloned corn genomic DNA, or amplified corn genomic DNA.

For instance, polymorphisms in DNA sequences can be detected byhybridization to allele-specific oligonucleotide (ASO) probes asdisclosed in U.S. Pat. Nos. 5,468,613 and 5,217,863. U.S. Pat. No.5,468,613 discloses allele specific oligonucleotide hybridizations wheresingle or multiple nucleotide variations in nucleic acid sequence can bedetected in nucleic acids by a process in which the sequence containingthe nucleotide variation is amplified, spotted on a membrane and treatedwith a labeled sequence-specific oligonucleotide probe.

Target nucleic acid sequence can also be detected by probe ligationmethods as disclosed in U.S. Pat. No. 5,800,944 where sequence ofinterest is amplified and hybridized to probes followed by ligation todetect a labeled part of the probe.

Microarrays can also be used for polymorphism detection, whereinoligonucleotide probe sets are assembled in an overlapping fashion torepresent a single sequence such that a difference in the targetsequence at one point would result in partial probe hybridization(Borevitz et al., Genome Res. 13:513-523 (2003); Cui et al.,Bioinformatics 21:3852-3858 (2005). On any one microarray, it isexpected there will be a plurality of target sequences, which mayrepresent genes and/or noncoding regions wherein each target sequence isrepresented by a series of overlapping oligonucleotides, rather than bya single probe. This platform provides for high throughput screening aplurality of polymorphisms. A single-feature polymorphism (SFP) is apolymorphism detected by a single probe in an oligonucleotide array,wherein a feature is a probe in the array. Typing of target sequences bymicroarray-based methods is disclosed in U.S. Pat. Nos. 6,799,122;6,913,879; and 6,996,476.

Target nucleic acid sequence can also be detected by probe linkingmethods as disclosed in U.S. Pat. No. 5,616,464 employing at least onepair of probes having sequences homologous to adjacent portions of thetarget nucleic acid sequence and having side chains which non-covalentlybind to form a stem upon base pairing of said probes to said targetnucleic acid sequence. At least one of the side chains has aphotoactivatable group which can form a covalent cross-link with theother side chain member of the stem.

Other methods for detecting SNPs and Indels include single baseextension (SBE) methods. Examples of SBE methods include, but are notlimited, to those disclosed in U.S. Pat. Nos. 6,004,744; 6,013,431;5,595,890; 5,762,876; and 5,945,283. SBE methods are based on extensionof a nucleotide primer that is immediately adjacent to a polymorphism toincorporate a detectable nucleotide residue upon extension of theprimer. In certain embodiments, the SBE method uses three syntheticoligonucleotides. Two of the oligonucleotides serve as PCR primers andare complementary to sequence of the locus of corn genomic DNA whichflanks a region containing the polymorphism to be assayed. Followingamplification of the region of the corn genome containing thepolymorphism, the PCR product is mixed with the third oligonucleotide(called an extension primer) which is designed to hybridize to theamplified DNA immediately adjacent to the polymorphism in the presenceof DNA polymerase and two differentially labeleddideoxynucleosidetriphosphates. If the polymorphism is present on thetemplate, one of the labeled dideoxynucleosidetriphosphates can be addedto the primer in a single base chain extension. The allele present isthen inferred by determining which of the two differential labels wasadded to the extension primer. Homozygous samples will result in onlyone of the two labeled bases being incorporated and thus only one of thetwo labels will be detected. Heterozygous samples have both allelespresent, and will thus direct incorporation of both labels (intodifferent molecules of the extension primer) and thus both labels willbe detected.

In a preferred method for detecting polymorphisms, SNPs and Indels canbe detected by methods disclosed in U.S. Pat. Nos. 5,210,015; 5,876,930;and 6,030,787 in which an oligonucleotide probe having a 5′fluorescentreporter dye and a 3′quencher dye covalently linked to the 5′ and 3′ends of the probe. When the probe is intact, the proximity of thereporter dye to the quencher dye results in the suppression of thereporter dye fluorescence, e.g. by Forster-type energy transfer. DuringPCR forward and reverse primers hybridize to a specific sequence of thetarget DNA flanking a polymorphism while the hybridization probehybridizes to polymorphism-containing sequence within the amplified PCRproduct. In the subsequent PCR cycle DNA polymerase with 5′ 3′exonuclease activity cleaves the probe and separates the reporter dyefrom the quencher dye resulting in increased fluorescence of thereporter.

Marker-Trait Associations

For the purpose of QTL mapping, the markers included should bediagnostic of origin in order for inferences to be made about subsequentpopulations. SNP markers are ideal for mapping because the likelihoodthat a particular SNP allele is derived from independent origins in theextant populations of a particular species is very low. As such, SNPmarkers are useful for tracking and assisting introgression of QTLs,particularly in the case of haplotypes.

The genetic linkage of additional marker molecules can be established bya gene mapping model such as, without limitation, the flanking markermodel reported by Lander et al., (Lander et al. 1989 Genetics,121:185-199), and the interval mapping, based on maximum likelihoodmethods described therein, and implemented in the software packageMAPMAKER/QTL (Lincoln and Lander, Mapping Genes Controlling QuantitativeTraits Using MAPMAKER/QTL, Whitehead Institute for Biomedical Research,Massachusetts, (1990). Additional software includes Qgene, Version 2.23(1996), Department of Plant Breeding and Biometry, 266 Emerson Hall,XXell University, Ithaca, N.Y.). Use of Qgene software is a particularlypreferred approach.

A maximum likelihood estimate (MLE) for the presence of a marker iscalculated, together with an MLE assuming no QTL effect, to avoid falsepositives. A log₁₀ of an odds ratio (LOD) is then calculated as:LOD=log₁₀ (MLE for the presence of a QTL/MLE given no linked QTL). TheLOD score essentially indicates how much more likely the data are tohave arisen assuming the presence of a QTL versus in its absence. TheLOD threshold value for avoiding a false positive with a givenconfidence, say 95%, depends on the number of markers and the length ofthe genome. Graphs indicating LOD thresholds are set forth in Lander etal. (1989), and further described by Arús and Moreno-González, PlantBreeding, Hayward, Bosemark, Romagosa (eds.) Chapman & Hall, London, pp.314-331 (1993).

Additional models can be used. Many modifications and alternativeapproaches to interval mapping have been reported, including the use ofnon-parametric methods (Kruglyak et al., 1995 Genetics, 139:1421-1428).Multiple regression methods or models can also be used, in which thetrait is regressed on a large number of markers (Jansen, Biometrics inPlant Breed, van Oijen, Jansen (eds.) Proceedings of the Ninth Meetingof the Eucarpia Section Biometrics in Plant Breeding, The Netherlands,pp. 116-124 (1994); Weber and Wricke, Advances in Plant Breeding,Blackwell, Berlin, 16 (1994)). Procedures combining interval mappingwith regression analysis, whereby the phenotype is regressed onto asingle putative QTL at a given marker interval, and at the same timeonto a number of markers that serve as ‘cofactors,’ have been reportedby Jansen et al. (Jansen et al., 1994 Genetics, 136:1447-1455) and Zeng(Zeng 1994 Genetics 136:1457-1468). Generally, the use of cofactorsreduces the bias and sampling error of the estimated QTL positions (Utzand Melchinger, Biometrics in Plant Breeding, van Oijen, Jansen (eds.)Proceedings of the Ninth Meeting of the Eucarpia Section Biometrics inPlant Breeding, The Netherlands, pp. 195-204 (1994), thereby improvingthe precision and efficiency of QTL mapping (Zeng 1994). These modelscan be extended to multi-environment experiments to analyzegenotype-environment interactions (Jansen et al., 1995 Theor. Appl.Genet. 91:33-3).

Selection of appropriate mapping populations is important to mapconstruction. The choice of an appropriate mapping population depends onthe type of marker systems employed (Tanksley et al., Molecular mappingin plant chromosomes. chromosome structure and function: Impact of newconcepts J. P. Gustafson and R. Appels (eds.). Plenum Press, New York,pp. 157-173 (1988)). Consideration must be given to the source ofparents (adapted vs. exotic) used in the mapping population. Chromosomepairing and recombination rates can be severely disturbed (suppressed)in wide crosses (adapted×exotic) and generally yield greatly reducedlinkage distances. Wide crosses will usually provide segregatingpopulations with a relatively large array of polymorphisms when comparedto progeny in a narrow cross (adapted×adapted).

An F₂ population is the first generation of selfing. Usually a single F₁plant is selfed to generate a population segregating for all the genesin Mendelian (1:2:1) fashion. Maximum genetic information is obtainedfrom a completely classified F₂ population using a codominant markersystem (Mather, Measurement of Linkage in Heredity: Methuen and Co.,(1938)). In the case of dominant markers, progeny tests (e.g. F₃, BCF₂)are required to identify the heterozygotes, thus making it equivalent toa completely classified F₂ population. However, this procedure is oftenprohibitive because of the cost and time involved in progeny testing.Progeny testing of F₂ individuals is often used in map constructionwhere phenotypes do not consistently reflect genotype (e.g. diseaseresistance) or where trait expression is controlled by a QTL.Segregation data from progeny test populations (e.g. F₃ or BCF₂) can beused in map construction. Marker-assisted selection can then be appliedto cross progeny based on marker-trait map associations (F₂, F₃), wherelinkage groups have not been completely disassociated by recombinationevents (i.e., maximum disequilibrium).

Recombinant inbred lines (RIL) (genetically related lines; usually >F₅,developed from continuously selfing F₂ lines towards homozygosity) canbe used as a mapping population. Information obtained from dominantmarkers can be maximized by using RIL because all loci are homozygous ornearly so. Under conditions of tight linkage (i.e., about <10%recombination), dominant and co-dominant markers evaluated in RILpopulations provide more information per individual than either markertype in backcross populations (Reiter et al., 1992 Proc. Natl. Acad.Sci. (USA) 89:1477-1481). However, as the distance between markersbecomes larger (i.e., loci become more independent), the information inRIL populations decreases dramatically.

Backcross populations (e.g., generated from a cross between a successfulvariety (recurrent parent) and another variety (donor parent) carrying atrait not present in the former) can be utilized as a mappingpopulation. A series of backcrosses to the recurrent parent can be madeto recover most of its desirable traits. Thus a population is createdconsisting of individuals nearly like the recurrent parent but eachindividual carries varying amounts or mosaic of genomic regions from thedonor parent. Backcross populations can be useful for mapping dominantmarkers if all loci in the recurrent parent are homozygous and the donorand recurrent parent have contrasting polymorphic marker alleles (Reiteret al., 1992). Information obtained from backcross populations usingeither codominant or dominant markers is less than that obtained from F₂populations because one, rather than two, recombinant gametes aresampled per plant. Backcross populations, however, are more informative(at low marker saturation) when compared to RILs as the distance betweenlinked loci increases in RIL populations (i.e. about 0.15%recombination). Increased recombination can be beneficial for resolutionof tight linkages, but may be undesirable in the construction of mapswith low marker saturation.

Near-isogenic lines (NIL) created by many backcrosses to produce anarray of individuals that are nearly identical in genetic compositionexcept for the trait or genomic region under interrogation can be usedas a mapping population. In mapping with NILs, only a portion of thepolymorphic loci are expected to map to a selected region.

Bulk segregant analysis (BSA) is a method developed for the rapididentification of linkage between markers and traits of interest(Michelmore et al., 1991 Proc. Natl. Acad. Sci. (U.S.A.) 88:9828-9832).In BSA, two bulked DNA samples are drawn from a segregating populationoriginating from a single cross. These bulks contain individuals thatare identical for a particular trait (resistant or susceptible toparticular disease) or genomic region but arbitrary at unlinked regions(i.e. heterozygous). Regions unlinked to the target region will notdiffer between the bulked samples of many individuals in BSA.

Marker-Assisted Breeding

Further, the present invention contemplates that preferred haploidplants comprising at least one genotype of interest are identified usingthe methods disclosed in U.S. Patent Application Ser. No. 60/837,864,which is incorporated herein by reference in its entirety, wherein agenotype of interest may correspond to a QTL or haplotype and isassociated with at least one phenotype of interest. The methods includeassociation of at least one haplotype with at least one phenotype,wherein the association is represented by a numerical value and thenumerical value is used in the decision-making of a breeding program.Non-limiting examples of numerical values include haplotype effectestimates, haplotype frequencies, and breeding values. In the presentinvention, it is particularly useful to identify haploid plants ofinterest based on at least one genotype, such that only those linesundergo doubling, which saves resources. Resulting doubled haploidplants comprising at least one genotype of interest are then advanced ina breeding program for use in activities related to germplasmimprovement.

In the present invention, haplotypes are defined on the basis of one ormore polymorphic markers within a given haplotype window, with haplotypewindows being distributed throughout the crop's genome. In anotheraspect, de novo and/or historical marker-phenotype association data areleveraged to infer haplotype effect estimates for one or more phenotypesfor one or more of the haplotypes for a crop. Haplotype effect estimatesenable one skilled in the art to make breeding decisions by comparinghaplotype effect estimates for two or more haplotypes. Polymorphicmarkers, and respective map positions, of the present invention areprovided in U.S. Patent Application Publication Nos 2005/0204780,2005/0216545, 2005/0218305, and Ser. No. 11/504,538, which areincorporated herein by reference in their entirety.

In yet another aspect, haplotype effect estimates are coupled withhaplotype frequency values to calculate a haplotype breeding value of aspecific haplotype relative to other haplotypes at the same haplotypewindow, or across haplotype windows, for one or more phenotypic traits.In other words, the change in population mean by fixing the haplotype isdetermined. In still another aspect, in the context of evaluating theeffect of substituting a specific region in the genome, either byintrogression or a transgenic event, haplotype breeding values are usedas a basis in comparing haplotypes for substitution effects. Further, inhybrid crops, the breeding value of haplotypes is calculated in thecontext of at least one haplotype in a tester used to produce a hybrid.Once the value of haplotypes at a given haplotype window are determinedand high density fingerprinting information is available on specificvarieties or lines, selection can be applied to these genomic regionsusing at least one marker in the at least one haplotype.

In the present invention, selection can be applied at one or more stagesof a breeding program:

a) Among genetically distinct populations, herein defined as “breedingpopulations,” as a pre-selection method to increase the selection indexand drive the frequency of favorable haplotypes among breedingpopulations, wherein pre-selection is defined as selection amongpopulations based on at least one haplotype for use as parents inbreeding crosses, and leveraging of marker-trait association identifiedin previous breeding crosses.b) Among segregating progeny from a breeding population, to increase thefrequency of the favorable haplotypes for the purpose of line or varietydevelopment.c) Among segregating progeny from a breeding population, to increase thefrequency of the favorable haplotypes prior to QTL mapping within thisbreeding population.d) For hybrid crops, among parental lines from different heteroticgroups to predict the performance potential of different hybrids.

In the present invention, it is contemplated that methods of determineassociations between genotype and phenotype in haploid plants can beperformed based on haplotypes, versus markers alone (Fan et al., 2006Genetics). A haplotype is a segment of DNA in the genome of an organismthat is assumed to be identical by descent for different individualswhen the knowledge of identity by state at one or more loci is the samein the different individuals, and that the regional amount of linkagedisequilibrium in the vicinity of that segment on the physical orgenetic map is high. A haplotype can be tracked through populations andits statistical association with a given trait can be analyzed. Bysearching the target space for a QTL association across multiple QTLmapping populations that have parental lines with genomic regions thatare identical by descent, the effective population size associated withQTL mapping is increased. The increased sample size results in morerecombinant progeny which increases the precision of estimating the QTLposition.

Thus, a haplotype association study allows one to define the frequencyand the type of the ancestral carrier haplotype. An “association study”is a genetic experiment where one tests the level of departure fromrandomness between the segregation of alleles at one or more marker lociand the value of individual phenotype for one or more traits.Association studies can be done on quantitative or categorical traits,accounting or not for population structure and/or stratification. In thepresent invention, associations between haplotypes and phenotypes forthe determination of “haplotype effect estimates” can be conducted denovo, using mapping populations for the evaluation of one or morephenotypes, or using historical genotype and phenotype data.

A haplotype analysis is important in that it increases the statisticalpower of an analysis involving individual biallelic markers. In a firststage of a haplotype frequency analysis, the frequency of the possiblehaplotypes based on various combinations of the identified biallelicmarkers of the invention is determined. The haplotype frequency is thencompared for distinct populations and a reference population. Ingeneral, any method known in the art to test whether a trait and agenotype show a statistically significant correlation may be used.

Methods for determining the statistical significance of a correlationbetween a phenotype and a genotype, in this case a haplotype, may bedetermined by any statistical test known in the art and with anyaccepted threshold of statistical significance being required. Theapplication of particular methods and thresholds of significance arewell within the skill of the ordinary practitioner of the art.

To estimate the frequency of a haplotype, the base reference germplasmhas to be defined (collection of elite inbred lines, population ofrandom mating individuals, etc.) and a representative sample (or theentire population) has to be genotyped. For example, in one aspect,haplotype frequency is determined by simple counting if considering aset of inbred individuals. In another aspect, estimation methods thatemploy computing techniques like the Expectation/Maximization (EM)algorithm are required if individuals genotyped are heterozygous at morethan one locus in the segment and linkage phase is unknown (Excoffier etal. 1995 Mol. Biol. Evol. 12: 921-927; Li et al., 2002 Biostatistics).Preferably, a method based on the EM algorithm (Dempster et al., 1977 J.R. Stat. Soc. Ser. B 39:1-38) leading to maximum-likelihood estimates ofhaplotype frequencies under the assumption of Hardy-Weinberg proportions(random mating) is used (Excoffier et al., 1995 Mol. Biol. Evol. 12:921-927). Alternative approaches are known in the art that forassociation studies: genome-wide association studies, candidate regionassociation studies and candidate gene association studies (Li et al.,2006 BMC Bioinformatics 7:258). The polymorphic markers of the presentinvention may be incorporated in any map of genetic markers of a plantgenome in order to perform genome-wide association studies.

The present invention comprises methods to detect an association betweenat least one haplotype in a haploid crop plant and a preferred trait,including a transgene, or a multiple trait index and calculate ahaplotype effect estimate based on this association. In one aspect, thecalculated haplotype effect estimates are used to make decisions in abreeding program. In another aspect, the calculated haplotype effectestimates are used in conjunction with the frequency of the at least onehaplotype to calculate a haplotype breeding value that will be used tomake decisions in a breeding program. A multiple trait index (MTI) is anumerical entity that is calculated through the combination of singletrait values in a formula. Most often calculated as a linear combinationof traits or normalized derivations of traits, it can also be the resultof more sophisticated calculations (for example, use of ratios betweentraits). This MTI is used in genetic analysis as if it were a trait.

Any given chromosome segment can be represented in a given population bya number of haplotypes that can vary from 1 (region is fixed), to thesize of the population times the ploidy level of that species (2 in adiploid species), in a population in which every chromosome has adifferent haplotype. Identity-by-descent among haplotype carried bymultiple individuals in a non-fixed population will result in anintermediate number of haplotype and possibly a differing frequencyamong the different haplotypes. New haplotypes may arise throughrecombination at meiosis between existing haplotypes in heterozygousprogenitors. The frequency of each haplotype may be estimated by severalmeans known to one versed in the art (e.g. by direct counting, or byusing an EM algorithm). Let us assume that “k” different haplotypes,identified as “h_(i)” (i=1, . . . , k), are known, that their frequencyin the population is “f_(i)” (i=1, . . . , k), and for each of thesehaplotypes we have an effect estimate “Est_(i)” (i=1, . . . , k). If wecall the “haplotype breeding value” (BV_(i)) the effect on thatpopulation of fixing that haplotype, then this breeding valuecorresponds to the change in mean for the trait(s) of interest of thatpopulation between its original state of haplotype distribution at thewindow and a final state at which haplotype “h_(i)” encounters itself ata frequency of 100%. The haplotype breeding value of h_(i) in thispopulation is calculated as:

${B\; V_{i}} = {{Est}_{i} - {\sum\limits_{i = 1}^{k}{{Est}_{i}f_{i}}}}$

One skilled in the art will recognize that haplotypes that are rare inthe population in which effects are estimated tend to be less preciselyestimated, this difference of confidence may lead to adjustment in thecalculation. For example one can ignore the effects of rare haplotypes,by calculating breeding value of better known haplotype after adjustingthe frequency of these (by dividing it by the sum of frequency of thebetter known haplotypes). One could also provide confidence intervalsfor the breeding value of each haplotypes.

The present invention anticipates that any particular haplotype breedingvalue will change according to the population for which it iscalculated, as a function of difference of haplotype frequencies. Theterm “population” will thus assume different meanings, below are twoexamples of special cases. In one aspect, a population is a singleinbred in which one intends to replace its current haplotype h_(j) by anew haplotype h_(i), in this case BV_(i)=Est_(i)−Est_(j). In anotheraspect, a “population” is a F2 population in which the two parentalhaplotype h_(i) and h_(j) are originally present in equal frequency(50%), in which case BV_(i)=½(Est_(i)−Est_(j)).

These statistical approaches enable haplotype effect estimates to informbreeding decisions in multiple contexts. Other statistical approaches tocalculate breeding values are known to those skilled in the art and canbe used in substitution without departing from the spirit and scope ofthis invention.

In cases where conserved genetic segments, or haplotype windows, arecoincident with segments in which QTL have been identified it ispossible to deduce with high probability that QTL inferences can beextrapolated to other germplasm having an identical haplotype in thathaplotype window. This a priori information provides the basis to selectfor favorable QTLs prior to QTL mapping within a given population. Forexample, plant breeding decisions could comprise:

a) Selection among haploid breeding populations to determine whichpopulations have the highest frequency of favorable haplotypes, whereinhaplotypes are designated as favorable based on coincidence withprevious QTL mapping and preferred populations undergo doubling; orb) Selection of haploid progeny containing the favorable haplotypes inbreeding populations prior to, or in substitution for, QTL mappingwithin that population, wherein selection could be done at any stage ofbreeding and at any generation of a selection and can be followed bydoubling; orc) Prediction of progeny performance for specific breeding crosses; ord) Selection of haploid plants for doubling for subsequent use ingermplasm improvement activities based on the favorable haplotypes,including line development, hybrid development, selection amongtransgenic events based on the breeding value of the haplotype that thetransgene was inserted into, making breeding crosses, testing andadvancing a plant through self fertilization, using plant or partsthereof for transformation, using plants or parts thereof for candidatesfor expression constructs, and using plant or parts thereof formutagenesis.

In cases where haplotype windows are coincident with segments in whichgenes have been identified it is possible to deduce with highprobability that gene inferences can be extrapolated to other germplasmhaving an identical genotype, or haplotype, in that haplotype window.This a priori information provides the basis to select for favorablegenes or gene alleles on the basis of haplotype identification within agiven population. For example, plant breeding decisions could comprise:

a) Selection among haploid breeding populations to determine whichpopulations have the highest frequency of favorable haplotypes, whereinhaplotypes are designated as favorable based on coincidence withprevious gene mapping and preferred populations undergo doubling; orb) Selection of haploid progeny containing the favorable haplotypes inbreeding populations, wherein selection is effectively enabled at thegene level, wherein selection could be done at any stage of breeding andat any generation of a selection and can be followed by doubling; orc) Prediction of progeny performance for specific breeding crosses; ord) Selection of haploid plants for doubling for subsequent use ingermplasm improvement activities based on the favorable haplotypes,including line development, hybrid development, selection amongtransgenic events based on the breeding value of the haplotype that thetransgene was inserted into, making breeding crosses, testing andadvancing a plant through self fertilization, using plant or partsthereof for transformation, using plants or parts thereof for candidatesfor expression constructs, and using plant or parts thereof formutagenesis.

A preferred haplotype provides a preferred property to a parent plantand to the progeny of the parent when selected by a marker means orphenotypic means. The method of the present invention provides forselection of preferred haplotypes, or haplotypes of interest, and theaccumulation of these haplotypes in a breeding population.

In the present invention, haplotypes and associations of haplotypes toone or more phenotypic traits provide the basis for making breedingdecisions and germplasm improvement activities. Non-limiting examples ofbreeding decisions include progeny selection, parent selection, andrecurrent selection for at least one haplotype. In another aspect,breeding decisions relating to development of plants for commercialrelease comprise advancing plants for testing, advancing plants forpurity, purification of sublines during development, inbred development,variety development, and hybrid development. In yet other aspects,breeding decisions and germplasm improvement activities comprisetransgenic event selection, making breeding crosses, testing andadvancing a plant through self-fertilization, using plants or partsthereof for transformation, using plants or parts thereof for candidatesfor expression constructs, and using plants or parts thereof formutagenesis.

In another embodiment, this invention enables indirect selection throughselection decisions for at least one phenotype based on at least onenumerical value that is correlated, either positively or negatively,with one or more other phenotypic traits. For example, a selectiondecision for any given haplotype effectively results in selection formultiple phenotypic traits that are associated with the haplotype.

In still another embodiment, the present invention acknowledges thatpreferred haplotypes identified by the methods presented herein may beadvanced as candidate genes for inclusion in expression constructs,i.e., transgenes. Nucleic acids underlying haplotypes of interest may beexpressed in plant cells by operably linking them to a promoterfunctional in plants. In another aspect, nucleic acids underlyinghaplotypes of interest may have their expression modified bydouble-stranded RNA-mediated gene suppression, also known as RNAinterference (“RNAi”), which includes suppression mediated by smallinterfering RNAs (“siRNA”), trans-acting small interfering RNAs(“ta-siRNA”), or microRNAs (“miRNA”). Examples of RNAi methodologysuitable for use in plants are described in detail in U.S. PatentApplication Publications 2006/0200878 and 2007/0011775.

Methods are known in the art for assembling and introducing constructsinto a cell in such a manner that the nucleic acid molecule for a traitis transcribed into a functional mRNA molecule that is translated andexpressed as a protein product. For the practice of the presentinvention, conventional compositions and methods for preparing and usingconstructs and host cells are well known to one skilled in the art, seefor example, Molecular Cloning: A Laboratory Manual, 3rd Edition Volumes1, 2, and 3 (2000) J. F. Sambrook, D. W. Russell, and N. Irwin, ColdSpring Harbor Laboratory Press. Methods for making transformationconstructs particularly suited to plant transformation include, withoutlimitation, those described in U.S. Pat. Nos. 4,971,908, 4,940,835,4,769,061 and 4,757,011, all of which are herein incorporated byreference in their entirety. Transformation methods for the introductionof expression units into plants are known in the art and includeelectroporation as illustrated in U.S. Pat. No. 5,384,253;microprojectile bombardment as illustrated in U.S. Pat. Nos. 5,015,580;5,550,318; 5,538,880; 6,160,208; 6,399,861; and 6,403,865; protoplasttransformation as illustrated in U.S. Pat. No. 5,508,184; andAgrobacterium-mediated transformation as illustrated in U.S. Pat. Nos.5,635,055; 5,824,877; 5,591,616; 5,981,840; and 6,384,301.

Another preferred embodiment of the present invention is to buildadditional value by selecting a composition of haplotypes wherein eachhaplotype has a haplotype effect estimate that is not negative withrespect to yield, or is not positive with respect to maturity, or isnull with respect to maturity, or amongst the best 50 percent withrespect to a phenotypic trait, transgene, and/or a multiple trait indexwhen compared to any other haplotype at the same chromosome segment in aset of germplasm, or amongst the best 50 percent with respect to aphenotypic trait, transgene, and/or a multiple trait index when comparedto any other haplotype across the entire genome in a set of germplasm,or the haplotype being present with a frequency of 75 percent or more ina breeding population or a set of germplasm provides evidence of itshigh value, or any combination of these.

This invention anticipates a stacking of haplotypes from multiplewindows into plants or lines by crossing parent plants or linescontaining different haplotype regions. The value of the plant or linecomprising in its genome stacked haplotype regions is estimated by acomposite breeding value, which depends on a combination of the value ofthe traits and the value of the haplotype(s) to which the traits arelinked. The present invention further anticipates that the compositebreeding value of a plant or line is improved by modifying thecomponents of one or each of the haplotypes. Additionally, the presentinvention anticipates that additional value can be built into thecomposite breeding value of a plant or line by selection of at least onerecipient haplotype with a preferred haplotype effect estimate or, inconjunction with the haplotype frequency, breeding value to which one orany of the other haplotypes are linked, or by selection of plants orlines for stacking haplotypes by breeding.

Another embodiment of this invention is a method for enhancing breedingpopulations by accumulation of one or more preferred haplotypes in a setof germplasm. Genomic regions defined as haplotype windows includegenetic information that contribute to one or more phenotypic traits ofthe plant. Variations in the genetic information at one or more loci canresult in variation of one or more phenotypic traits, wherein the valueof the phenotype can be measured. The genetic mapping of the haplotypewindows allows for a determination of linkage across haplotypes. Ahaplotype of interest has a DNA sequence that is novel in the genome ofthe progeny plant and can in itself serve as a genetic marker for thehaplotype of interest. Notably, this marker can also be used as anidentifier for a gene or QTL. For example, in the event of multipletraits or trait effects associated with the haplotype, only one markerwould be necessary for selection purposes. Additionally, the haplotypeof interest may provide a means to select for plants that have thelinked haplotype region. Selection can be performed by screening fortolerance to an applied phytotoxic chemical, such as an herbicide orantibiotic, or to pathogen resistance. Selection may be performed usingphenotypic selection means, such as, a morphological phenotype that iseasy to observe such as seed color, seed germination characteristic,seedling growth characteristic, leaf appearance, plant architecture,plant height, and flower and fruit morphology.

The present invention also provides for the screening of progeny haploidplants for haplotypes of interest and using haplotype effect estimatesas the basis for selection for use in a breeding program to enhance theaccumulation of preferred haplotypes. The method includes: a) providinga breeding population comprising at least two haploid plants wherein thegenome of the breeding population comprises a plurality of haplotypewindows and each of the plurality of haplotype windows comprises atleast one haplotype; and b) associating a haplotype effect estimate forone or more traits for two or more haplotypes from one or more of theplurality of haplotype windows, wherein the haplotype effect estimatecan then be used to calculate a breeding value that is a function of theestimated effect for any given phenotypic trait and the frequency ofeach of the at least two haplotypes; and c) ranking one or more of thehaplotypes on the basis of a value, wherein the value is a haplotypeeffect estimate, a haplotype frequency, or a breeding value and whereinthe value is the basis for determining whether a haplotype is apreferred haplotype, or haplotype of interest; and d) utilizing theranking as the basis for decision-making in a breeding program; and e)at least one progeny haploid plant is selected for doubling on the basisof the presence of the respective markers associated with the haplotypesof interest, wherein the progeny haploid plant comprises in its genomeat least a portion of the haplotype or haplotypes of interest of thefirst plant and at least one preferred haplotype of the second plant;and f) using resulting doubled haploid plants in activities related togermplasm improvement wherein the activities are selected from the groupconsisting of line and variety development, hybrid development,transgenic event selection, making breeding crosses, testing andadvancing a plant through self fertilization, using plant or partsthereof for transformation, using plants or parts thereof for candidatesfor expression constructs, and using plant or parts thereof formutagenesis.

Using this method, the present invention contemplates that haplotypes ofinterest are selected from a large population of plants, and theselected haplotypes can have a synergistic breeding value in thegermplasm of a crop plant. Additionally, this invention provides forusing the selected haplotypes in the described breeding methods toaccumulate other beneficial and preferred haplotype regions and to bemaintained in a breeding population to enhance the overall germplasm ofthe crop plant.

Plant Breeding

Plants of the present invention can be part of or generated from abreeding program. The choice of breeding method depends on the mode ofplant reproduction, the heritability of the trait(s) being improved, andthe type of cultivar used commercially (e.g., F₁ hybrid cultivar,pureline cultivar, etc). A cultivar is a race or variety of a plantspecies that has been created or selected intentionally and maintainedthrough cultivation.

Selected, non-limiting approaches for breeding the plants of the presentinvention are set forth below. A breeding program can be enhanced usingmarker assisted selection (MAS) on the progeny of any cross. It isunderstood that nucleic acid markers of the present invention can beused in a MAS (breeding) program. It is further understood that anycommercial and non-commercial cultivars can be utilized in a breedingprogram. Factors such as, for example, emergence vigor, vegetativevigor, stress tolerance, disease resistance, branching, flowering, seedset, seed size, seed density, standability, and threshability etc. willgenerally dictate the choice.

Genotyping can be further economized by high throughput, non-destructiveseed sampling. In one embodiment, plants can be screened for one or moremarkers, such as genetic markers, using high throughput, non-destructiveseed sampling. In a preferred aspect, haploid seed is sampled in thismanner and only seed with at least one marker genotype of interest isadvanced for doubling. Apparatus and methods for the high-throughput,non-destructive sampling of seeds have been described which wouldovercome the obstacles of statistical samples by allowing for individualseed analysis. For example, U.S. patent application Ser. No. 11/213,430(filed Aug. 26, 2005); U.S. patent application Ser. No. 11/213,431(filed Aug. 26, 2005); U.S. patent application Ser. No. 11/213,432(filed Aug. 26, 2005); U.S. patent application Ser. No. 11/213,434(filed Aug. 26, 2005); and U.S. patent application Ser. No. 11/213,435(filed Aug. 26, 2005), U.S. patent application Ser. No. 11/680,611(filed Mar. 2, 2007), which are incorporated herein by reference intheir entirety, disclose apparatus and systems for the automatedsampling of seeds as well as methods of sampling, testing and bulkingseeds.

For highly heritable traits, a choice of superior individual plantsevaluated at a single location will be effective, whereas for traitswith low heritability, selection should be based on mean values obtainedfrom replicated evaluations of families of related plants. Popularselection methods commonly include pedigree selection, modified pedigreeselection, mass selection, and recurrent selection. In a preferredaspect, a backcross or recurrent breeding program is undertaken.

The complexity of inheritance influences choice of the breeding method.Backcross breeding can be used to transfer one or a few favorable genesfor a highly heritable trait into a desirable cultivar. This approachhas been used extensively for breeding disease-resistant cultivars.Various recurrent selection techniques are used to improvequantitatively inherited traits controlled by numerous genes.

Breeding lines can be tested and compared to appropriate standards inenvironments representative of the commercial target area(s) for two ormore generations. The best lines are candidates for new commercialcultivars; those still deficient in traits may be used as parents toproduce new populations for further selection.

The development of new elite corn hybrids requires the development andselection of elite inbred lines, the crossing of these lines andselection of superior hybrid crosses. The hybrid seed can be produced bymanual crosses between selected male-fertile parents or by using malesterility systems. Additional data on parental lines, as well as thephenotype of the hybrid, influence the breeder's decision whether tocontinue with the specific hybrid cross.

Pedigree breeding and recurrent selection breeding methods can be usedto develop cultivars from breeding populations. Breeding programscombine desirable traits from two or more cultivars or variousbroad-based sources into breeding pools from which cultivars aredeveloped by selfing and selection of desired phenotypes. New cultivarscan be evaluated to determine which have commercial potential.

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous cultivaror inbred line, which is the recurrent parent. The source of the traitto be transferred is called the donor parent. After the initial cross,individuals possessing the phenotype of the donor parent are selectedand repeatedly crossed (backcrossed) to the recurrent parent. Theresulting plant is expected to have most attributes of the recurrentparent (e.g., cultivar) and, in addition, the desirable traittransferred from the donor parent.

The single-seed descent procedure in the strict sense refers to plantinga segregating population, harvesting a sample of one seed per plant, andusing the one-seed sample to plant the next generation. When thepopulation has been advanced from the F₂ to the desired level ofinbreeding, the plants from which lines are derived will each trace todifferent F₂ individuals. The number of plants in a population declineseach generation due to failure of some seeds to germinate or some plantsto produce at least one seed. As a result, not all of the F₂ plantsoriginally sampled in the population will be represented by a progenywhen generation advance is completed.

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (Allard, “Principles of Plant Breeding,” John Wiley & Sons, NY, U.of CA, Davis, Calif., 50-98, 1960; Simmonds, “Principles of CropImprovement,” Longman, Inc., NY, 369-399, 1979; Sneep and Hendriksen,“Plant Breeding Perspectives,” Wageningen (ed), Center for AgriculturalPublishing and Documentation, 1979; Fehr, In: Soybeans: Improvement,Production and Uses, 2nd Edition, Manograph., 16:249, 1987; Fehr,“Principles of Variety Development,” Theory and Technique, (Vol. 1) andCrop Species Soybean (Vol. 2), Iowa State Univ., Macmillan Pub. Co., NY,360-376, 1987).

An alternative to traditional QTL mapping involves achieving higherresolution by mapping haplotypes, versus individual markers (Fan et al.,2006 Genetics 172:663-686). This approach tracks blocks of DNA known ashaplotypes, as defined by polymorphic markers, which are assumed to beidentical by descent in the mapping population. This assumption resultsin a larger effective sample size, offering greater resolution of QTL.Methods for determining the statistical significance of a correlationbetween a phenotype and a genotype, in this case a haplotype, may bedetermined by any statistical test known in the art and with anyaccepted threshold of statistical significance being required. Theapplication of particular methods and thresholds of significance arewell with in the skill of the ordinary practitioner of the art.

It is further understood, that the present invention provides bacterial,viral, microbial, insect, mammalian and plant cells comprising thenucleic acid molecules of the present invention.

As used herein, a “nucleic acid molecule,” be it a naturally occurringmolecule or otherwise may be “substantially purified”, if desired,referring to a molecule separated from substantially all other moleculesnormally associated with it in its native state. More preferably asubstantially purified molecule is the predominant species present in apreparation. A substantially purified molecule may be greater than 60%free, preferably 75% free, more preferably 90% free, and most preferably95% free from the other molecules (exclusive of solvent) present in thenatural mixture. The term “substantially purified” is not intended toencompass molecules present in their native state.

The agents of the present invention will preferably be “biologicallyactive” with respect to either a structural attribute, such as thecapacity of a nucleic acid to hybridize to another nucleic acidmolecule, or the ability of a protein to be bound by an antibody (or tocompete with another molecule for such binding). Alternatively, such anattribute may be catalytic, and thus involve the capacity of the agentto mediate a chemical reaction or response.

The agents of the present invention may also be recombinant. As usedherein, the term recombinant means any agent (e.g. DNA, peptide etc.),that is, or results, however indirect, from human manipulation of anucleic acid molecule.

The agents of the present invention may be labeled with reagents thatfacilitate detection of the agent (e.g. fluorescent labels (Prober etal. 1987 Science 238:336-340; Albarella et al., European Patent 144914),chemical labels (Sheldon et al., U.S. Pat. No. 4,582,789; Albarella etal., U.S. Pat. No. 4,563,417), modified bases (Miyoshi et al. EuropeanPatent 119448).

Having now generally described the invention, the same will be morereadily understood through reference to the following examples which areprovided by way of illustration, and are not intended to be limiting ofthe present invention, unless specified.

EXAMPLES Example 1 Phenotyping for GLS Reaction

In order to detect QTL associated with GLS resistance, plants werephenotyped to determine GLS reaction. The following rating scale wasused for phenotypic rating for GLS was used in all studies. Thepercentage of leaf area infected is used to rate plants on a scale of 1(very resistant) to 9 (susceptible). Disease resistance is evaluatedvisually after pollination. The infection can be natural or byartificial inoculation in the experiments.

TABLE 1 Description of rating scale used for GLS phenotyping.Description Rating Symptoms Very Resistant 1 0% of leaf area infected;no visible lesions Very Resistant 2 ILA<1%; few lesions, dispersedthrough lower leaves Resistant 3 1% ≦ ILA ≦ 20% Resistant 4 20% ≦ ILA ≦40% Mid-resistant 5 40% ≦ ILA ≦ 50%; lesions reaching ear leaf, withspare lesions in the leaves above the ear Mid-Susceptible 6 50% ≦ ILA ≦60%; lesions reaching the leaves above the ear Susceptible 7 60% ≦ ILA ≦75% Susceptible 8 75% ≦ ILA ≦ 90% Susceptible 9 >90% of foliar areainfected, with premature death of the plant before forming black layerILA = infected leaf area.

Example 2 GLS Resistance Mapping Study 1

To examine associations between SNP markers and GLS resistance in corn,analyzed data from a number of studies was combined. An associationstudy was conducted to evaluate whether significant associations betweenone or more marker genotypes and GLS resistance are present in one ormore breeding crosses. The mapping study combined data from 176 mappingpopulations. The number of individuals in each population ranged from 95to 276. Segregating populations were of the following generations F2,BC1F2, BC1, and DH. The number of SNP markers used for genotyping rangedfrom 55 to 158. Individuals were phenotyped for traits, including GLSresistance. A total of 2499 associations between SNP markers and GLSresistance were identified on Chromosomes 1, 2, 3, 4, 5, 6, 7, 8, 9, and10. The SNP markers provided can be used to monitor the introgression ofGLS resistance into a breeding population. SNP markers associated withGLS resistance, level of significance, and favorable alleles arereported in FIG. 1.

Example 3 GLS Resistance Mapping Study 2

An association study was conducted to evaluate whether significantassociations between one or more marker genotypes and GLS resistance arepresent in one or more breeding crosses. In the association study, 769F2s from the CV128/CV162 population were screened with 117 markers. Atotal of 53 associations between SNP markers and GLS resistance wereidentified on Chromosomes 1, 2, 3, 4, 5, 6, and 8. The SNP markersprovided can be used to monitor the introgression of GLS resistance intoa breeding population. SNP markers associated with GLS resistance, levelof significance, and favorable alleles are reported in FIG. 1.

Example 4 GLS Resistance Mapping Study 3

An association study was conducted to evaluate whether significantassociations between one or marker genotypes and GLS resistance arepresent in one or more populations. In the association study, 1177inbred corn lines were screened with 1051 SNP markers. A total of 92significant associations between SNP markers and GLS resistance wereidentified on Chromosomes 5, 6, 7, 8, 9, and 10. The SNP markersprovided can be used to monitor the introgression of GLS resistance intoa breeding population. SNP markers associated with GLS resistance, levelof significance, and favorable alleles are reported in FIG. 1.

Example 5 GLS Resistance Mapping Study 4

An association study was conducted to evaluate whether significantassociations between one or marker genotypes and GLS resistance arepresent in one or more populations. In this association study, 1036 DHlines from 398 F1 families were screened with 2,136 SNP markers. A totalof 205 significant associations between SNP markers and GLS resistancewere identified on Chromosomes 1, 2, 3, 4, 5, 6, 7, 8, 8, and 10. TheSNP markers provided can be used to monitor the introgression of GLSresistance into a breeding population. SNP markers associated with GLSresistance, level of significance, and favorable alleles are reported inFIG. 1.

Example 6 GLS Resistance Mapping Study 5

An association study was conducted to evaluate whether significantassociations between one or more marker genotypes and GLS resistance arepresent in one or more populations. In this association study, 495Single seed descent (SSD) lines from 495 F1 families were screened with1958 SNP markers. A total of 309 significant associations between SNPmarkers and GLS resistance were identified on Chromosomes 1, 2, 3, 4, 5,6, 7, 8, 9, and 10. The SNP markers provided can be used to monitor theintrogression of GLS resistance into a breeding population. SNP markersassociated with GLS resistance, level of significance, and favorablealleles are reported in FIG. 1.

From the association studies of Examples 2 through 6, 1227 SNP markerswere found to be associated with GLS. QTL were assigned by dividingmaize chromosomal regions into 10 cM windows. A total of 176 QTL wereidentified by associating SNP markers with GLS resistance. The favorablealleles used for selecting for GLS resistance are also provided inFIG. 1. Selection for GLS resistance is based on the genotype of GLSresistant parent.

Example 7 Exemplary Marker Assays for Detecting GLS Resistance

In one embodiment, the detection of polymorphic sites in a sample ofDNA, RNA, or cDNA may be facilitated through the use of nucleic acidamplification methods. Such methods specifically increase theconcentration of polynucleotides that span the polymorphic site, orinclude that site and sequences located either distal or proximal to it.Such amplified molecules can be readily detected by gel electrophoresis,fluorescence detection methods, or other means. Exemplary primers andprobes for amplifying and detecting genomic regions associated with GLSresistance are given in Table 2.

TABLE 2 Exemplary assays for detecting GLS resistance. SEQ ID SEQ IDMarker SNP Forward Reverse SEQ ID SEQ ID Marker SEQ ID Position PrimerPrimer Probe 1 Probe 2 NC0199588 1219 137 1304 1305 1306 1307 NC0055894421 202 1308 1309 1310 1311 NC0028145 481 307 1312 1313 1314 1315NC0003425 1127 280 1316 1317 1318 1319

Example 8 Oligonucleotide Hybridization Probes Useful for Detecting CornPlants with GLS Resistance Loci

Oligonucleotides can also be used to detect or type the polymorphismsassociated with GLS resistance disclosed herein by hybridization-basedSNP detection methods. Oligonucleotides capable of hybridizing toisolated nucleic acid sequences which include the polymorphism areprovided. It is within the skill of the art to design assays withexperimentally determined stringency to discriminate between the allelicstate of the polymorphisms presented herein. Exemplary assays includeSouthern blots, Northern blots, microarrays, in situ hybridization, andother methods of polymorphism detection based on hybridization.Exemplary oligonucleotides for use in hybridization-based SNP detectionare provided in Table 3. These oligonucleotides can be detectablylabeled with radioactive labels, fluorophores, or other chemiluminescentmeans to facilitate detection of hybridization to samples of genomic oramplified nucleic acids derived from one or more corn plants usingmethods known in the art.

TABLE 3 Exemplary Oligonucleotide Hybridization Probes*. SEQ ID SNPHybridization SEQ ID Marker Marker Position Probe Probe NC0199588 1219137 CAGCGCAGGGCTAGCT 1320 NC0199588 1219 137 CAGCGCAGAGCTAGCT 1321NC0055894 421 202 CCCAGTCGCAGTCCTA 1322 NC0055894 421 202CCCAGTCGTAGTCCTA 1323 NC0028145 481 307 ACAGCAACAAACCCAA 1324 NC0028145481 307 ACAGCAACGAACCCAA 1325 NC0003425 1127 280 ATGTGCCTGGTACCAG 1326NC0003425 1127 280 ATGTGCCTCGTACCAG 1327 *SNP nucleotides in bold.

Example 9 Oligonucleotide Probes Useful for Detecting Corn Plants withGLS Resistance Loci by Single Base Extension Methods

Oligonucleotides can also be used to detect or type the polymorphismsassociated with GLS resistance disclosed herein by single base extension(SBE)-based SNP detection methods. Exemplary oligonucleotides for use inSBE-based SNP detection are provided in Table 4. SBE methods are basedon extension of a nucleotide primer that is hybridized to sequencesimmediately adjacent to a polymorphism to incorporate a detectablenucleotide residue upon extension of the primer. It is also anticipatedthat the SBE method can use three synthetic oligonucleotides. Two of theoligonucleotides serve as PCR primers and are complementary to thesequence of the locus which flanks a region containing the polymorphismto be assayed. Exemplary PCR primers that can be used to type certainpolymorphisms disclosed in this invention are provided in Table 3 in thecolumns labeled “Forward Primer SEQ ID” and “Reverse Primer SEQ ID”.Following amplification of the region containing the polymorphism, thePCR product is hybridized with an extension primer which anneals to theamplified DNA immediately adjacent to the polymorphism. DNA polymeraseand two differentially labeled dideoxynucleoside triphosphates are thenprovided. If the polymorphism is present on the template, one of thelabeled dideoxynucleoside triphosphates can be added to the primer in asingle base chain extension. The allele present is then inferred bydetermining which of the two differential labels was added to theextension primer. Homozygous samples will result in only one of the twolabeled bases being incorporated and thus only one of the two labelswill be detected. Heterozygous samples have both alleles present, andwill thus direct incorporation of both labels (into different moleculesof the extension primer) and thus both labels will be detected.

TABLE 4 Probes (extension primers) for SingleBase Extension (SBE) assays. SNP Marker Posi- Probe Marker SEQ ID tionProbe (SBE) SEQ ID NC0199588 1219 137 ATCGACGATCAGCGCAG 1328 NC0055894421 202 GACACGGTTCCCAGTCG 1329 NC0028145 481 307 TACATATGCACAGCAAC 1330NC0003425 1127 280 ACATGTGACATGTGCCT 1331

Example 10 Fine Mapping for GLS Resistance

Three populations were developed for associating marker genotypes andGLS resistance. GLS resistant donor lines CV174 and CV173 were eachbackcrossed three times to I294213 to create backcross mappingpopulations. An additional population was developed using CV171 as theresistant source. CV171 was backcrossed two times to I294213 and selfedone generation for fine mapping. Composite interval mapping wasconducted. SNP markers associated with GLS resistance are provided inTable 5.

TABLE 5 SNP markers associated with GLS resistance. Fav Favorable SNPSEQ QTL Marker Chr pos LOD Effect Parent Allele Position ID 116NC0009667 6 139.1 6.966013 0.738745 CV171 G 226 883 83 NC0053636 6 1366.896417 0.749228 CV171 A 202 882 117 NC0032368 6 144.3 6.2265230.709636 CV171 G 801 1360 116 NC0002782 6 133.5 6.194592 0.858607 CV171C 121 881 38 NC0108013 2 115.3 5.835505 0.734241 CV171 C 340 306 37NC0151288 2 107.6 5.69125 0.868826 CV171 A 1001 303 115 NC0003201 6127.9 3.889942 0.780583 CV171 G 74 875 38 NC0035094 2 116.9 6.760.666924 CV174 G 173 310 77 NC0002474 4 93.6 6.02 0.674649 CV173 C 383571 170 NC0040011 10 54.2 2 0.503823 CV173 A 598 1361 65 NC0009079 3194.2 1.82 0.443853 CV173 C 118 484 82 NC0038447 4 141.8 1.79 0.651293CV173 A 526 618 89 NC0105613 5 16.6 1.59 0.375097 CV173 G 178 667 29NC0107911 2 99.2 1.51 0.396756 CV173 T 384 289 7 NC0009159 1 66 1.490.423507 CV173 A 360 56 128 NC0015161 7 106.4 0.64 0.253679 CV173 G 428962 156 NC0055759 9 62.1 0.54 0.287342 CV173 G 149 1100 148 NC0008757 8156.3 0.16 0.312206 CV173 C 274 1075

Example 11 Haploid Mapping Study for GLS Resistance with I133314/I206447Population

The utility of haploid plants in genetic mapping of traits of interestis demonstrated in the following example. A haploid population wasdeveloped by crossing the inbred corn lines I133314 by I206447 and theninducing the resulting F1 hybrid to produce 1945 haploid plants. Formapping, 82 SNP markers were used to screen the haploid population.Phenotypic data relating to GLS reaction were collected on thepopulation. Composite interval mapping was conducted to examinesignificant associations between GLS and the SNP markers. Table 6provides the significant marker associations found in this study. QTLassociated with GLS resistance were identified by genetic mapping withhaploid plants. The source of the favorable allele for GLS resistancewas I206447 for all markers except NC0151453 (SEQ ID NO: 1231) in whichthe source of the favorable allele was I133314. The chromosome (Chr.)location, chromosome position (Chr. pos), and favorable allele areprovided for each marker in Table 6.

It is appreciated by one skilled in the art that haploid plants can begenerated from any generation of plant population and that the methodsof the present invention can be used with one or more individuals,including SSD, from any generation of plant population. Non-limitingexamples of plant populations include F1, F2, BC1, BC2F1, F3:F4, F2:F3,and so on, including subsequent filial generations, as well asexperimental populations such as RILs and NILs. It is furtheranticipated that the degree of segregation within the one or more plantpopulations of the present invention can vary depending on the nature ofthe trait and germplasm under evaluation.

TABLE 6 Markers useful for detecting QTL associated with GLS in theI133314/I206447 haploid mapping population. Chr. Favorable SEQ ID SNPMarker Chr. position GLS QTL LOD Effect Allele Marker Position*NC0147103 1 39.1 177 6.15 0.17 C 1228 1001 NC0202383 2 19 2 20.38 0.30 T1229 34 NC0201657 2 179.2 178 26.30 0.34 T 1230 342 NC0055894 3 112.4 576.74 0.17 T 421 202 NC0151453 6 75.1 110 17.17 −0.28 T 1231 119 *SNPposition: refers to position of the SNP polymorphism in the indicatedSEQ ID NO.

Example 12 Haploid Mapping Study for GLS Resistance with I294213/I283669Population

The utility of haploid plants in genetic mapping of traits of interestis demonstrated in the following example. A haploid mapping populationwas developed by crossing the inbred corn lines I294213 by I283669. Theresulting F1 hybrid was induced to produce 1895 haploid seed. Formapping, 82 SNP markers were used to screen the haploid population.Composite interval mapping was conducted to examine significantassociations between GLS and the SNP markers. Table 8 provides thesignificant marker associations found in this study. QTL associated withGLS resistance were identified by genetic mapping with haploid plants.The source of the favorable alleles was I283669 for all makers exceptNC0003425 (SEQ ID NO: 1127) in which the source of the favorable allelewas I294213. The chromosome (Chr.) location, chromosome position (Chr.pos), and favorable allele are provided for each marker in Table 7.

It is appreciated by one skilled in the art that the methods of thepresent invention can be used with one or more individuals, includingSSD, from any generation of plant population. Non-limiting examples ofplant populations include to F1, F2, BC1, BC2F1, F3:F4, F2:F3, and soon, including subsequent filial generations, as well as experimentalpopulations such as RILs and NILs. It is further anticipated that thedegree of segregation within the one or more plant populations of thepresent invention can vary depending on the nature of the trait andgermplasm under evaluation.

TABLE 7 Markers useful for detecting QTL associated with GLS resistancein the I294213/I283669 haploid mapping population. Favo- SEQ SNP GLSrable ID Posi- Marker Chr pos QTL LOD Effect Allele Marker tionNC0052741 1 49.5 5 136.19 0.75 G 36 411 NC0028145 3 187.5 64 3.19 0.10 G481 307 NC0143354 5 1.8 88 8.33 0.16 C 659 303 NC0040408 6 59.1 10813.29 0.22 T 1232 336 NC0109097 7 93.8 127 5.93 0.13 T 1233 97 NC00034259 84.5 158 20.30 −0.25 G 1127 280 NC0199588 10 99.9 173 15.36 0.23 G1219 137 * SNP Position: refers to the position of the SNP polymorphismin the indicated SEQ ID NO.

Example 13 Introgression of GLS Resistance in Breeding

Given the description of the above-described GLS resistance loci, anillustrative example is presented for the utility of said GLS resistanceloci in a corn breeding program and, more specifically, in the contextof development of inbred lines. GLS resistant line CV171 is used as adonor source. Corn inbred CV009 is used as the recurrent parent. Table 8provides exemplary SNP markers and favorable alleles for selecting GLSresistant lines. Exemplary SNP markers NC0019588, NC0037947, NC0088767,NC0059114, NC0003201, NC0060514, NC0002782, NC0053636, NC0009667, andNC0032368 (SEQ ID NOs: 858, 860, 862, 866, 875, 877, 881, 882, 883, and1360) are used to monitor introgression of GLS resistance regions fromChromosome 6. A breeder selects for lines carrying the resistance allelefor one or more of said SNP markers, representing one or more GLSresistance loci.

The introgression of one or more resistance loci is achieved viarepeated backcrossing to a recurrent parent accompanied by selection toretain one or more GLS resistance loci from the donor parent using theabove-described markers. This backcross procedure is implemented at anystage in line development and occurs in conjunction with breeding forsuperior agronomic characteristics or one or more traits of interest,including transgenic and nontransgenic traits.

Alternatively, a forward breeding approach is employed wherein one ormore GLS resistance loci can be monitored for successful introgressionfollowing a cross with a susceptible parent with subsequent generationsgenotyped for one or more GLS resistance loci and for one or moreadditional traits of interest, including transgenic and nontransgenictraits.

TABLE 8 SNP markers useful for introgression of GLS resistance frominbred CV171 Marker SEQ Favorable Marker ID NO. Chromosome alleleNC0019588 858 6 C NC0037947 860 6 G NC0088767 862 6 A NC0059114 866 6 TNC0003201 875 6 G NC0060514 877 6 CA NC0002782 881 6 C NC0053636 882 6 ANC0009667 883 6 G NC0032368 1360 6 G

In view of the foregoing, it will be seen that the several advantages ofthe invention are achieved and attained.

The embodiments were chosen and described in order to best explain theprinciples of the invention and its practical application to therebyenable others skilled in the art to best utilize the invention invarious embodiments and with various modifications as are suited to theparticular use contemplated.

Various patent and non-patent publications are cited herein, thedisclosures of each of which are, to the extent necessary, incorporatedherein by reference in their entireties.

As various modifications could be made in the constructions and methodsherein described and illustrated without departing from the scope of theinvention, it is intended that all matter contained in the foregoingdescription or shown in the accompanying drawings shall be interpretedas illustrative rather than limiting. The breadth and scope of thepresent invention should not be limited by any of the above-describedexemplary embodiments, but should be defined only in accordance with thefollowing claims appended hereto and their equivalents.

We claim:
 1. A method of introgressing a Gray Leaf Spot (GLS) resistanceQTL allele into a corn plant, the method comprising the steps of: a)crossing at least one first corn plant comprising the Grey Leaf Spotresistance QTL allele, wherein the allele comprises SEQ ID NO: 1048,with at least one second corn plant in order to form a segregatingpopulation; b) screening said segregating population with one or morenucleic acid markers to determine if one or more corn plants from saidsegregating population contain the Gray Leaf Spot resistance QTL allelecomprising SEQ ID NO: 1048; and c) selecting from said segregatingpopulation one or more corn plants comprising said allele associatedwith a Gray Leaf Spot (GLS) resistance QTL.
 2. A method of creating apopulation of corn plants comprising at least one allele associated withGray Leaf Spot (GLS) resistance comprising SEQ ID NO: 1048, the methodcomprising the steps of: a) genotyping a first population of cornplants, said population containing at least one allele associated withGray Leaf Spot resistance, the at least one allele associated with GreyLeaf Spot resistance comprising SEQ ID NO: 1048; b) selecting from saidfirst population of corn plants based upon said genotyping one or moreidentified corn plants containing said at least one allele associatedwith Grey Leaf Spot resistance comprising SEQ ID NO: 1048; and c)growing from said selected corn plants a second population of cornplants, thereby creating a population of corn plants comprising at leastone allele associated with Grey Leaf Spot resistance comprising SEQ IDNO:
 1048. 3. The method according to claim 2, wherein the firstpopulation of corn plants genotyped in step (a) is a populationgenerated by a cross.
 4. The method of claim 3, wherein said cross iseffected by mechanical emasculation, chemical sterilization, or geneticsterilization of a pollen acceptor.
 5. The method of claim 2, whereingenotyping is effected in step (a) by determining the allelic state ofat least one corn genomic DNA marker.
 6. The method according to claim2, wherein the selected one or more identified corn plants exhibit atleast partial resistance to a GLS-inducing fungus or at leastsubstantial resistance to a GLS-inducing fungus.
 7. The method of claim3, wherein said first population of corn plants is generated by a crossof at least one Gray Leaf Spot (GLS) resistant corn plant with at leastone Gray Leaf Spot (GLS) sensitive corn plant.
 8. The method of claim 3,wherein said first population of corn plants is a segregatingpopulation.
 9. The method of claim 3, wherein said cross is a back crossof at least one Gray Leaf Spot (GLS) resistant corn plant with at leastone Gray Leaf Spot (GLS) sensitive corn plant to introgress GLSresistance into a corn germplasm.
 10. The method of claim 3, whereinsaid first population of corn plants is a haploid breeding population.11. The method according to claim 1, wherein at least one of the one ormore nucleic acid markers is located within 5 cM of SEQ ID NO:
 1048. 12.The method according to claim 11, wherein at least one of the one ormore nucleic acid markers is located within 2 cM of SEQ ID NO:
 1048. 13.The method according to claim 12, wherein at least one of the one ormore nucleic acid markers is located within 1 cM of SEQ ID NO:
 1048. 14.The method according to claim 1, wherein at least one of the one or morenucleic acid markers exhibits a LOD score of greater than 4.0 with saidGray Leaf Spot (GLS) resistance QTL allele.
 15. The method according toclaim 1, wherein said population is generated by a cross of at least oneGray Leaf Spot (GLS) resistant corn plant with at least one Gray LeafSpot (GLS) sensitive corn plant.
 16. The method of claim 1, wherein saidpopulation is a haploid breeding population.
 17. The method of claim 1,wherein said one or more nucleic acid markers is SEQ ID NO: 1048.